RAPID PUBLICATION
Production of Tumor Necrosis Factor with Sensitized Lung Tissue
19B Receptor Triggering from
Isao Ohno, Yuichi Ohkawara, Kohei Yamauchi, Yasuo Tanno, and Tamotsu Takishima The First Department of Internal Medicine, Tohoku University School of Medicine, Sendai, Japan
Mast cells and basophils have been known to playa central role in allergic inflammation through the release of chemical mediators by cross-linkage of IgE receptors. The IgE receptor triggering and calcium ionophore A23187 have also been shown to induce gene expression and production of tumor necrosis factor (TNF) by rat basophilic leukemia cells. In the present study, we examined whether IgE receptor triggering could induce gene expression and production of TNF in rat lung tissue. The lung tissue released not only histamine but also cytotoxic activity on L929 cells 2 and 4 h after incubation with dinitrophenyl conjugated to ovalbumin (DNP-OVA) following passive sensitization with anti-DNP monoclonal rat IgE antibody, whereas neither DNP-OVA nor anti-DNP IgE antibody could induce the cytotoxic activity when used solely. Calcium ionophore A23187 also could induce both histamine release and cytotoxic activity. These activities induced by IgE receptor triggering, A23187, and lipopolysaccharide were completely neutralized by preincubation with anti-mouse TNF-rabbit serum, but not with normal rabbit serum. Northern blot analysis using cDNA probe of mouse TNF demonstrated expression of TNF gene as early as 2 h after IgE receptor triggering. These data demonstrating that IgE receptor triggering induced gene expression and production of TNF in lung tissue suggest the participation of TNF in the pathogenesis of late asthmatic response through its biologic activities such as the attraction and activation of neutrophils and eosinophils.
Mast cells and basophils have been shown to play a major role in immediate allergic reaction by releasing chemical mediators such as histamine, serotonin, and arachidonate metabolites after the triggering of IgE receptor with specific antigen (1). However, it has also been reported that, in response to the cross-linkage of IgE receptor, cultured mast cells not only released chemical mediators but also produced various cytokines such as interleukin (IL)-3, IL-4, IL-5, IL-6, and granulocyte/macrophage colony-stimulating factor (2, 3). Moreover, tumor necrosis factor (TNF), which is a cytokine produced mainly by monocytes and macrophages after stimulation with lipopolysaccharide (LPS) (4), and a TNFlike factor were demonstrated to be released from mast cells or basophils. Young and associates showed that, after stimu-
Key Words: tumor necrosis factor, IgE receptor, lung tissue, bronchial asthma (Received in original form July 10, 1990 and in revised form July 26, 1990) Address correspondence to: Tamotsu Takishima, M.D., Professor and Chairman, The First Department of Internal Medicine, 1-1 Seiryo-Machi, Aoba-ku, Sendai 980, Japan. Abbreviations: dinitropheny1, DNP: dinitrophenyl-ovalbumin, DNP-OVA; interleukin, IL; lipopolysaccharide, LPS; mouse tumor necrosis factor, mTNF; phorbol myristate acetate, PMA; tumor necrosis factor, TNF. Am. J. Respir. CeU Mol. BioI. Vol. 3. pp. 285-289, 1990
lation with phorbol myristate acetate (PMA) or LPS, murine mast cells produced a TNF-like factor whose activity was partially blocked by anti-TNF antibody, and that the molecular weight of the factor was 50 kD in sodium dodecyl sulfate-polyacrylamide gel electrophoresis under reducing conditions, unlike reported TNF (5). Mast cells were reported to express TNF mRNA under conditions of no stimulation (6) or after stimulation with PMA (7). Recently, we demonstrated that antigen challenge following passive sensitization induced not only histamine release but also gene expression and production of TNF molecule in rat basophilic leukemia cells (8). On the other hand, TNF is well known to display a broad spectrum of biologic properties such as chemotaxis and activation ofneutrophils (9-11) and eosinophils (12, 13), and the expression of adhesion molecule for neutrophils on endothelial cells (14, 15), in addition to its direct tumoricidal activities (16, 17), causes it to playa pivotal role in the enhancement of inflammation. These studies suggest that TNF is produced by mast cells or basophils after stimulation with antigen and contributes to lung inflammation such as asthmatic response initiated by the stimulation of these cells with specific antigen. However, the release of TNF from lung tissue after antigen challenge has not been observed yet. Therefore, we evaluated gene expression and production of TNF in rat lung tissue that was passively sensitized with monoclonal IgE antibody followed by stimulation with specific antigen.
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AMERICAN JOURNAL OF RESPIRATORY CELL AND MOLECULAR BIOLOGY VOL. 3 1990
Materials and Methods Preparation of Lung Tissue Male Sprague-Dawley rats, 200 to 250 g, purchased from Shizuoka Laboratory Animal Center (Hamamatsu, Japan) were killed, and the lung was perfused via the aorta with distilled saline to avoid blood cell contamination, until the lung tissue appeared uniformly white. Approximately 2 to 3 g of total lung was extracted, and, after resection of the trachea, minced finely with sterilized scissors. The resulting fragments (about 10 mg each) were washed twice with Eagle's minimal essential medium (MEM) (GIBCO Laboratories, Grand Island, NY), with 100 U/ml penicillin (Meiji Seika Kaisha Ltd., Tokyo, Japan) and 100 p.g/ml streptomycin (Meiji). These procedures were performed in tissue culture dishes (100 X 20 rom style; Becton Dickinson Labware, Lincoln Park, NJ) on ice. It took about 2 h for the culture of lung tissue to start.
Histamine release (%)
=
S
X
100/(S
+ L).
Spontaneous release was obtained from the experiment in which the fragments were incubated with medium alone and was less than 10% of the total histamine content. Experimental results were expressed as the percentage ofhistamine release after stimulation from which that of spontaneous release was subtracted. Cytotoxic Assay for TNF The cytotoxic activity of the culture supernatants was measured on L929 cells with actinomycin D (Sigma) (8). Units were defined as the reciprocal of the dilution of the supernatant at which 50 % cytotoxicity was obtained, and the cytotoxic activity was expressed as units per gram of wet lung. The activity was expressed as 0 when it was less than 2 U, which was the limitation in this system. In addition, some supernatants and recombinant mouse TNF (mTNF) (> 99% pure,4 X 107 U/mg on L929 cells; Genzyme Corporation, Boston, MA) were assayed in the presence of a neutralizing rabbit antiserum to recombinant mTNF (approximately 1()6 neutralizing U/ml; Genzyme) or normal rabbit serum (TAGO Inc., Burlingame, CA) at 1,000-fold dilution.
Culture of Lung Fragments Approximately 200 to 300 mg of the lung fragments were incubated with 1 ml of LPS (Escherichia coli 026:B6; Sigma Chemical Co., St. Louis, MO) at 10 p.g/ml, calcium ionophore A23187 (Sigma) at 2.5 p.g/ml, or medium alone in a well of 12-well tissue culture plates (Costar, Cambridge, MA). For IgE receptor triggering, the fragments were at first passively sensitized with 10% of anti-dinitrophenyl (DNP) monoclonal rat IgE in MEM, which was incubated at 56° C for 4 h before passive sensitization in some experiments, for 2 h in tissue culture dishes (35 x 10 rom style; Corning Glass Works, Corning, NY). Anti-DNP monoclonal rat IgE was kindly provided by Creative Products Research Laboratories (Kissei Pharmaceutical Co. Ltd., Matsumoto, Japan) (8). After sensitization, the fragments were washed with MEM sufficiently, and cultured with DNP conjugated to ovalbumin (DNP-OVA) or anti-rat IgE monoclonal mouse IgG (Serotec, Kidlington, UK) as mentioned above. The experiments were performed in duplicate in a 12-well culture plate, and the plates were incubated in the moist chamber gassed with a mixture of 40% O2 and room air at 37° C. After varying incubation periods, the culture supernatants and fragments were harvested. Constituents oftissue culture media were screened for endotoxin contamination using the colorimetric assay of Toxicolor test (Seikagaku Kogyo Co. Ltd., Tokyo, Japan). Contamination of endotoxin was less than 40 pg/ml, which was shown to be unable to stimulate macrophages to produce TNF (18).
Histamine Release from Rat Lung Tissue When the lung fragments were incubated with DNP-OVA at concentrations of 0.01 to 100 p.g/ml after passive sensitization, histamine was released from the fragments dosedependently. Histamine was maximally released after stimulation with 100 p.g/ml of DNP-OVA (12.8 ± 5.1%, n = 3) (Figure 1). When the passive sensitization was performed with medium alone, DNP-OVA at any concentration could not release histamine significantly compared to spontaneously released histamine (data not shown).
Analysis of Histamine Release Approximately 100 mg of the lung fragments with or without passive sensitization were incubated with 0.5 ml of DNPOVA, anti-rat IgE antibody or A23187 at various concentrations in a well of 24-well culture plates (Becton Dickinson) in duplicate. After 1 h, in each well, the histamine content of the culture supernatants (S) and the remaining histamine (L) extracted by boiling the lung fragments for 10 min in phosphate-buffered saline acidified by addition of 12 N HCI (Sigma) to pH 3 to 4 were measured with an automated fluorometric technique (19). The percentage of histamine release in each well was calculated using the following formula:
Cytotoxic Activity in Culture Supernatants of Lung Fragments Culture supernatants of lung fragments were harvested after 1, 2, or 4 h of stimulation (Figure 2). The stimulation with LPS resulted in the release of a high level of cytotoxic activity (279.2 ± 86.4 U/g wet lung, n = 4) after 4 h, and a slight activity (10.2 ± 5.7, n = 3) was detected 4 h after stimulation with A23187, which induced the release of histamine (15.3 ± 6.9). Moreover, the sensitized fragments released the cytotoxic activity 2 and 4 h after stimulation with DNPOVA (44.8 ± 14.6, n = 3 and 106.9 ± 41.5, n = 3, respectively), whereas the incubation of the sensitized fragments
RNA Preparation and Northern Blot Analysis After incubation for various periods, lung fragments were homogenized in 5.2 M guanidine isothiocyanate solution (20). As previously described (8), RNA was extracted and hybridized with mTNF cDNA (provided by Asahi Chemical Industry Co. Ltd.) (21) labeled with (32P]dCTP-a (Amersham International, Buckinghamshire, UK). Statistical Analysis Data were expressed as the mean ± SEM, and statistical significance was assessed using Student's t test.
Results
Ohno, Ohkawara, Yamauchi et al.: IgE Receptor-mediated TNF Production
287
20
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Ol
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-
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o
100
Figure 1. Histamine release from rat lung tissue with specific antigen. After sensitization, lung fragments were incubated with DNPOVA at concentrations of 0.01 to 100 Itg/ml for 1 h. Experimental results (mean ± SEM, n = 3) were expressed as the percentage of histamine release as described in MATERIAUi AND METHODS.
with medium alone induced the release of only a small level of the cytotoxic activity after 4 h (1.5 ± 1.5, n = 4). On the other hand, the fragments that were passively sensitized with either medium alone or heat-inactivated anti-DNP IgE antibody failed to release cytotoxic activity, even if stimulated with DNP-OVA (data not shown). The incubation with DNPOVA following the passive sensitization with anti-DNP monoclonal IgE has been demonstrated to be able to stimulate IgE receptors on basophils (8). These data suggested that the release of the cytotoxic activity from the lung fragments after stimulation with specific antigen was not due to contamination of endotoxin but to the cross-linkage of IgE receptors. This was confirmed by the evidence that incubation with anti-rat IgE antibody at IOO-fold dilution could induce not only the release of histamine but also that of cytotoxic activity from the lung fragments that were passively sensitized with anti-DNP rat monoclonal IgE antibody, but not from the lung fragments that were passively sensitized with medium alone (data not shown). Neutralization of the Cytotoxic Activity by Anti-mTNF Specific Antibody Anti-mTNF antibody abolished the cytotoxic activity in the culture medium of the lung fragments stimulated with LPS as well as that of recombinant mTNF on L929 cells. In addition, either the cytotoxic activity from the sensitized lung fragments stimulated with DNP-OVA or that from the fragments stimulated with A23187 was completely neutralized after the incubation with anti-mTNF antibody (Table 1). These data suggested that these activities obtained from lung
•
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"T
234
1
Time Ch) Figure 2. Kinetics of cytotoxic activity from rat lung tissue. Lung fragments were incubated with A23187 at 5 Itg/ml (triangles), or DNP-OVA at 100 Itg/ml (circles), or medium alone (crosses) following passive sensitization. After 1, 2, and 4 h, the culture supernatants were assayed for cytotoxic activity and expressed as units per gram of wet lung (mean ± SEM, n = 3 to 4). After stimulation with LPS at 10 Itg/ml for 4 h, 279.2 ± 86.4 (n = 4) of cytotoxic activity was obtained from lung fragments.
fragments had the same immunoreactivity to anti-mTNFspecific serum as mTNF. TNF Gene Expression in Lung Fragments To evaluate TNF gene expression, we hybridized mRNA extracted from rat lung fragments with mTNF eDNA probe (Figure 3). The probe hybridized with RNA from the lung fragments stimulated with LPS. In addition, the hybridized bands with the probe were observed as early as 2 h after IgE receptor triggering at the same position as that after stimula-
TABLE 1
Neutralization of cytotoxic activity by anti-mTNF rabbit serum* % Residual Cytotoxicity after Treatment with: Sample
Lung tissue +LPS +DNP-OVA +A23187 mTNF
Normal Serum
87.3 93.3 76.4 98.0
± ± ± ±
4.4 2.5 8.6 0.9
Anti-mTNF
0.0 0.0 0.3 0.8
± ± ± ±
0.0 0.0 0.3 0.8
* Cultured supernatants of lung fragments and recombinant mTNF were assayed for cytotoxicity after incubation with anti-mTNF rabbit serum as described in MATERIALS AND METHODS. Residual cytotoxicity was calculated according to the following formula: (cytotoxicity with treatment/cytotoxicity without treatment) x 100. Data are expressed as mean ± SEM (n = 4).
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AMERICAN JOURNAL OF RESPIRATORY CELL AND MOLECULAR BIOLOGY VOL. 3 1990
Medium
o
2
4
+DNP-OVA
8h 2
4
8h
+LPS
2h
285185-
Figure 3. TNF gene expression in rat lung tissue. RNA was extracted at indicated time points after stimulation with medium, DNP-OVA (100 JLg/ml) following passive sensitization, or LPS (10 JLg/ml), and loaded at 20 JLg to each lane. The nitrocellulose filter was hybridized with 32P-labeled mTNF cDNA probe. The migration positions of28S and 18S ribosomal RNA were indicated to the left. An equal amount of RNA in each lane was confirmed with the assessment of 28S and 18S ribosomal RNA stained with acrigine orange after electrophoresis.
tion with LPS, while not after medium alone. An equal amount of RNA in each lane was confirmed with the assessment of 28S and 18S ribosomal RNA stained with acrigine orange after electrophoresis.
Discussion We have demonstrated that the incubation with a specific antigen or anti-IgE antibody following passive sensitization with monoclonal IgE antibody induced not only histamine release but also the gene expression and production of TNF in cultured lung tissue. The contamination of endotoxin in the reagents used in this study was less than 40 pg/ml, which was unable to induce detectable TNF production from macrophages (18). Either specific antigen, anti-IgE antibody, or IgE antibody failed to induce the release of both histamine and cytotoxic activity from the lung fragments when used solely. The passive sensitization with heat-inactivated antiDNP IgE antibody could not induce the release of cytotoxic activity after stimulation with DNP-OVA. These results suggested that IgE receptor-mediated stimulation induced the release of cytotoxic activity from lung tissue. Several laboratories have provided evidence of TNF-like factors in mast cells (5-7). In rat basophilic leukemia cells, we previously showed that the gene expression and production of TNF was induced in response to the cross-linkage ofIgE receptors (8). Recently, using an organ culture of human skin, Klein and colleagues (22) reported that anti-IgE, calcium ionophore, and morphine sulfate induced degranulation of mast cells and expression of an endothelial molecule responsible for leukocyte adhesion, and that the induction of the molecule might be mediated by TNF because anti-human TNF blocked the expression of the molecule. They discussed that mast cells might release a factor with TNF-like properties, by the observation that preincubation with cromolyn sodium, which is an inhibitor of mast cell degranulation, abrogated
the expression of the adherent molecule, though TNF-producing cells were not identified. In our experiments, lung mast cells are the possible cellular source of TNF, and directly express TNF gene transcripts and produce TNF molecules in response to IgE receptor triggering. However, TNF is produced mainly by activated monocytes and macrophages (4), which are also known to express low affinity Fete) receptors (23) and to participate as effector cells in the pathology of allergic reactions through the release of leukotrienes (24), prostaglandins (25), platelet-activating factor (26), and IL-l (27) through IgE receptor triggering. Therefore, it is possible to suggest that macrophages in lung tissue directly release TNF through IgE-mediated stimulation. In addition, there are various types of stromal cells in normal lung tissue such as endothelial cells, vascular smooth muscle cells, epithelial cells, and fibroblasts. Cultured human vascular smooth muscle cells express TNF gene and release TNF protein after stimulation with LPS (28), and TNF mRNA was shown to localize in murine intestinal epithelium (29). These observations raise the possibility that the indirect stimulation of stromal cells through activation of mast cells or macrophages by IgE receptor triggering results in the gene expression and production of TNF in these cells. A23187, which is known to be able to stimulate macrophages to produce TNF (30) and mast cells/basophils to release histamine (31), was shown to induce gene expression and production of TNF in rat basophilic leukemia cells (8) and to activate endothelial cells through the induction of TNF in human skin culture (22). However, we could not determine which cells, macrophages, mast cells, or other types of cells, released TNF after stimulation with A23187. IgE receptor triggering by specific antigen followed by mast cell degranulation play a central role in the pathogenesis of bronchial asthma (1), and, in whole lung tissue studies, it has been observed that chemical mediators such as histamine and leukotrienes were released after antigen challenge (32, 33). However, we demonstrated not only histamine release but also the upregulation of TNF gene and the release of a factor immunologically related to TNF from lung tissue after IgE receptor triggering. Our data represent the first description of cytokine production by whole lung tissue with IgE receptor triggering. On the other hand, it has become clear that TNF contributes indirectly to the recruitment and activation of inflammatory cells such as monocytes, neutrophils, and eosinophils, suggesting that they might attribute to the pathogenesis of late response of bronchial asthma after allergen challenge (34). Therefore, our results may imply the important role ofTNF in the inflammation of bronchial asthma after antigen challenge. Acknowledgments: The writers thank Mrs. Yuko Satoh for technical assistance. This work was supported in part by a Grant-in-Aid for Scientific Research from the Ministry of Education, Science and Culture (Japan).
References I. Wasserman, S. I. 1979. The mast cell and the inflammatory response. In . The Mast Cell: Its Role in Health and Disease. J. Pepys and A. M. Edwards, editors. Pitman Medical Publishing Co., Tumbridge Wells, UK. 9-20. 2. Wodnar-Filipowicz, A., C. H. Heusser, and C. Moroni. 1989. Production of haemopoietic growth factors GM-CSF and interleukin-3 by mast cells in response to IgE receptor-mediated activation. Nature 339:150-152. 3. Plaut, M., J. H. Pierce, C. J. Watson, J. Hanley-Hyde, R. P. Nordan, and
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W. E. Paul. 1989. Mast cell lines produce lymphokines in response to cross-linkage of Fe RI or to calcium ionophores. Nature 339:64-67. Beutler, B., J. Mahonery, N. Le Trang, P. Pekala, and A. Cerami. 1985. Purification of cachectin, a lipoprotein lipase-suppressing hormone secreted by endotoxin-induced RAW 264.7 cells. J. Exp. Med. 161: 984-995. Young, J. D., C. Liu, G. Butler, Z. A. Cohn, and S. J. Galli. 1987. Identification, purification, and characterization of a mast cell-associated cytolytic factor related to tumor necrosis factor. Proc. Natl. Acad. Sci. USA 84:9175-9179. Steffen, M., M. Abboud, G. K. Potter, Y. P. Yung, and M. A. S. Moore. 1989. Presence of tumor necrosis factor or a related factor in human basophil/mast cells. Immunology 66:445-450. Ohno, I., Y. Tanno, K. Yamauchi, and T. Takishima. 1990. Production of tumor necrosis factor by mastocytoma P815 cells. Immunology 69: 312-315. Ohno, I., Y. Tanno, K. Yamauchi, and T. Takishima. 1990. Gene expression and production of tumor necrosis factor by a rat basophilic leukemia cell line (RBL-2H3) with IgE receptor triggering. Immunology 70:88-93. Ming, W. J., L. Bersani, and A. Mantovani. 1987. Tumor necrosis factor is chemotactic for monocytes and polymorphonuclear leukocytes. J. Immunol. 138: 1469-1474. Klebanoff, S. J., M. A. Vadas, J. M. Harlan et al. 1986. Stimulation of neutrophils by tumor necrosis factor. J. Immunol. 136:4220-4225. Shalaby, M. R., B. B. Aggarwal, E. Rinderknecht, L. P. Svedersky, B. S. Finkle, and M. A. Palladino Jr. 1985. Activation of human polymorphonuclear neutrophil functions by interferon-y and tumor necrosis factors. J. Immunol. 135:2069-2073. Silberstein, D. S., and J. R. David. 1986. Tumor necrosis factor enhances eosinophil toxicity to Schistosoma mansoni larvae. Proc. Natl. Acad. Sci. USA 83:1055-1059. Lamas, A. M., C. M. Mulroney, and R. P. Schleimer. 1988. Studies on the adhesive interaction between purified human eosinophils and cultured vascular endothelial cells. J. Immunol. 140:1500-1505. Rothlein, R., M. Czajkowski, M. M. O'Neill, S. D. Marlin, E. Mainofi, and V. J. Merluzzi. 1988. Induction of intercellular adhesion molecule 1 on primary and continuous cell lines by proinflanunatory cytokines. Regulation by pharmacologic agents and neutralizing antibodies. J. Immunol. 141:1665-1669. Pober, J. S., M. P. Bevilacqua, D. L. Mendrick, L. A. Lapierre, W. Fiers, and M. A. Gimbrone. 1986. Two distinct monokines, interleukin 1 and tumor necrosis factor, each independently induce biosynthesis and transient expression of the same antigen on the surface of cultured human vascular endothelial cells. J. Immunol. 136:1680-1687. Sugarman, B. J., B. B. Aggarwal, P. E. Hass, I. S. Figari, M. A. Palladino Jr., and H. M. Shepard. 1985. Recombinant human tumor necrosis factor-a: effects on proliferation of normal and transformed cells in vitro. Science 230:943-945. Ruggiero, V., K. Latham, and C. Baglioni. 1987. Cytostatic and cytotoxic activity of tumor necrosis factor on human cancer cells. J. Immunol. 138:2711-2717. Weinberg, 1. B., H. A. Chapman, Jr., and 1. B. Hibbs, Jr. 1978. Characterization of the effects of endotoxin on macrophage tumor cell killing. J. Immunol. 121:72-80.
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19. Siraganian, R. P., and W. A. Hook. 1980. Histamine release and assay methods for the study of human allergy. In Manual of Clinical Immunology. N. E. Rose and H. Friedman, editors. American Society for Microbiology, Washington, DC. 808-821. 20. Chirgwin,1. M., A. E. Przybyla, R. J. MacDonald, and W. 1. Rutter. 1979. Isolation of biologically active ribonucleic acid from sources enriched in ribonuclease. Biochemistry 18:5294-5299. 21. Shirai, T., N. Shimizu, S. Shiojiri, S. Horiguchi, and H. Ito. 1988. Cloning and expression in Escherichia coli of the gene for mouse tumor necrosis factor. DNA 7:193-201. 22. Klein, L. M., R. M. Lavker, W. L. Matis, andG. F. Murphy. 1989. Degranulation of human mast cells induces an endothelial antigen central to leukocyte adhesion. Proc. Natl. Acad. Sci. USA 86:8972-8976. 23. Capron, A., J. P. Dessaint, M. Capron, M. Joseph, J. C. Ameisen, and A. B. Tonnel. 1986. From parasites to allergy: a second receptor for IgE. Immunology Today 7:15-18. 24. Ferreri, N. R., R. S. Zeiger, and H. L. Spiegelberg. 1988. IgG-, IgA-, and IgE-induced release of leukotriene C4 by monocytes isolated from patients with atopic dermatitis. J. Allergy Clin. Immunol. 82:556-567. 25. Rouzer, C. A., W. A. Scott, A. L. Hamill, F.-T. Liu, D. H. Katz, and Z. A. Cohn. 1982. Secretion of leukotriene C and other archidonic acid metabolites by macrophages challenged with immunoglobulin E immune complexes. J. Exp. Med. 156:1077-1086. 26. Joseph, M., A.-B. Tonnel, G. Torpier, and A. Capron. 1983. Involvement of immunoglobulin E in the secretory processes of alveolar macrophages from asthmatic patients. J. Clin. Invest. 71:221-230. 27. Mazingue, C., V. Carriere, J.-P. Dessaint et al. 1987. Regulation of IgE synthesis by macrophages expressing Fee-receptors: role of interleukin 1. Clin. Exp. Immunol. 67:587-593. 28. Warner, S. J. C., and P. Libby. 1989. Human vascular smooth muscle cells. Target for and source of tumor necrosis factor. J. Immunol. 142: 100-109. 29. Keshav, S., L. Lawson, L. P. Chung, M. Stein, V. H. Perry, and S. Gordon. 1990. Tumor necrosis factor mRNA localized to Paneth cells of normal murine intestinal epithelium by in situ hybridization. J. Exp. Med. 171:327-332. 30. Zacharchuk, C. M., B.-E. Drysdale, M. M. Mayer, and H. S. Shin. 1983. Macrophage-mediated cytotoxicity: role of a soluble macrophage cytotoxic factor similar to Iymphotoxin and tumor necrosis factor. Proc. Natl. Acad. Sci. USA 80:6341-6345. 31. Behrendt, H., U. Rosenkranz, and W. Schmutzler. 1978. Ultrastructure of isolated human mast cells during histamine release induced by ionophore A23187. Int. Arch. Allergy Appl. Immunol. 56:188-192. 32. Sheard, P., P. G. Killingback, and A. M. J. N. Blair. 1967. Antigen induced release of histamine and SRS-A from human lung passively sensitized with reaginic serum. Nature 216:283-284. 33. Schulman, E. S., H. H. Newball, L. M. Demers, F. A. Fitzpatrick, and N. F. Adkinson Jr. 1981. Anaphylactic release of thromboxane A 2 , prostaglandin D" and prostacyclin from human lung parenchyma. Am. Rev. Respir. Dis. 124:402-406. 34. Schleimer, R. P. 1990. Effects of glucocorticosteroids on inflammatory cells relevant to their therapeutic applications in asthma. Am. Rev. Respir. Dis. 141:S59-S69.
Production of Tumor Necrosis Factor with Sensitized Lung Tissue
19B Receptor Triggering from
Isao Ohno, Yuichi Ohkawara, Kohei Yamauchi, Yasuo Tanno, and Tamotsu Takishima The First Department of Internal Medicine, Tohoku University School of Medicine, Sendai, Japan
Mast cells and basophils have been known to playa central role in allergic inflammation through the release of chemical mediators by cross-linkage of IgE receptors. The IgE receptor triggering and calcium ionophore A23187 have also been shown to induce gene expression and production of tumor necrosis factor (TNF) by rat basophilic leukemia cells. In the present study, we examined whether IgE receptor triggering could induce gene expression and production of TNF in rat lung tissue. The lung tissue released not only histamine but also cytotoxic activity on L929 cells 2 and 4 h after incubation with dinitrophenyl conjugated to ovalbumin (DNP-OVA) following passive sensitization with anti-DNP monoclonal rat IgE antibody, whereas neither DNP-OVA nor anti-DNP IgE antibody could induce the cytotoxic activity when used solely. Calcium ionophore A23187 also could induce both histamine release and cytotoxic activity. These activities induced by IgE receptor triggering, A23187, and lipopolysaccharide were completely neutralized by preincubation with anti-mouse TNF-rabbit serum, but not with normal rabbit serum. Northern blot analysis using cDNA probe of mouse TNF demonstrated expression of TNF gene as early as 2 h after IgE receptor triggering. These data demonstrating that IgE receptor triggering induced gene expression and production of TNF in lung tissue suggest the participation of TNF in the pathogenesis of late asthmatic response through its biologic activities such as the attraction and activation of neutrophils and eosinophils.
Mast cells and basophils have been shown to play a major role in immediate allergic reaction by releasing chemical mediators such as histamine, serotonin, and arachidonate metabolites after the triggering of IgE receptor with specific antigen (1). However, it has also been reported that, in response to the cross-linkage of IgE receptor, cultured mast cells not only released chemical mediators but also produced various cytokines such as interleukin (IL)-3, IL-4, IL-5, IL-6, and granulocyte/macrophage colony-stimulating factor (2, 3). Moreover, tumor necrosis factor (TNF), which is a cytokine produced mainly by monocytes and macrophages after stimulation with lipopolysaccharide (LPS) (4), and a TNFlike factor were demonstrated to be released from mast cells or basophils. Young and associates showed that, after stimu-
Key Words: tumor necrosis factor, IgE receptor, lung tissue, bronchial asthma (Received in original form July 10, 1990 and in revised form July 26, 1990) Address correspondence to: Tamotsu Takishima, M.D., Professor and Chairman, The First Department of Internal Medicine, 1-1 Seiryo-Machi, Aoba-ku, Sendai 980, Japan. Abbreviations: dinitropheny1, DNP: dinitrophenyl-ovalbumin, DNP-OVA; interleukin, IL; lipopolysaccharide, LPS; mouse tumor necrosis factor, mTNF; phorbol myristate acetate, PMA; tumor necrosis factor, TNF. Am. J. Respir. CeU Mol. BioI. Vol. 3. pp. 285-289, 1990
lation with phorbol myristate acetate (PMA) or LPS, murine mast cells produced a TNF-like factor whose activity was partially blocked by anti-TNF antibody, and that the molecular weight of the factor was 50 kD in sodium dodecyl sulfate-polyacrylamide gel electrophoresis under reducing conditions, unlike reported TNF (5). Mast cells were reported to express TNF mRNA under conditions of no stimulation (6) or after stimulation with PMA (7). Recently, we demonstrated that antigen challenge following passive sensitization induced not only histamine release but also gene expression and production of TNF molecule in rat basophilic leukemia cells (8). On the other hand, TNF is well known to display a broad spectrum of biologic properties such as chemotaxis and activation ofneutrophils (9-11) and eosinophils (12, 13), and the expression of adhesion molecule for neutrophils on endothelial cells (14, 15), in addition to its direct tumoricidal activities (16, 17), causes it to playa pivotal role in the enhancement of inflammation. These studies suggest that TNF is produced by mast cells or basophils after stimulation with antigen and contributes to lung inflammation such as asthmatic response initiated by the stimulation of these cells with specific antigen. However, the release of TNF from lung tissue after antigen challenge has not been observed yet. Therefore, we evaluated gene expression and production of TNF in rat lung tissue that was passively sensitized with monoclonal IgE antibody followed by stimulation with specific antigen.
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AMERICAN JOURNAL OF RESPIRATORY CELL AND MOLECULAR BIOLOGY VOL. 3 1990
Materials and Methods Preparation of Lung Tissue Male Sprague-Dawley rats, 200 to 250 g, purchased from Shizuoka Laboratory Animal Center (Hamamatsu, Japan) were killed, and the lung was perfused via the aorta with distilled saline to avoid blood cell contamination, until the lung tissue appeared uniformly white. Approximately 2 to 3 g of total lung was extracted, and, after resection of the trachea, minced finely with sterilized scissors. The resulting fragments (about 10 mg each) were washed twice with Eagle's minimal essential medium (MEM) (GIBCO Laboratories, Grand Island, NY), with 100 U/ml penicillin (Meiji Seika Kaisha Ltd., Tokyo, Japan) and 100 p.g/ml streptomycin (Meiji). These procedures were performed in tissue culture dishes (100 X 20 rom style; Becton Dickinson Labware, Lincoln Park, NJ) on ice. It took about 2 h for the culture of lung tissue to start.
Histamine release (%)
=
S
X
100/(S
+ L).
Spontaneous release was obtained from the experiment in which the fragments were incubated with medium alone and was less than 10% of the total histamine content. Experimental results were expressed as the percentage ofhistamine release after stimulation from which that of spontaneous release was subtracted. Cytotoxic Assay for TNF The cytotoxic activity of the culture supernatants was measured on L929 cells with actinomycin D (Sigma) (8). Units were defined as the reciprocal of the dilution of the supernatant at which 50 % cytotoxicity was obtained, and the cytotoxic activity was expressed as units per gram of wet lung. The activity was expressed as 0 when it was less than 2 U, which was the limitation in this system. In addition, some supernatants and recombinant mouse TNF (mTNF) (> 99% pure,4 X 107 U/mg on L929 cells; Genzyme Corporation, Boston, MA) were assayed in the presence of a neutralizing rabbit antiserum to recombinant mTNF (approximately 1()6 neutralizing U/ml; Genzyme) or normal rabbit serum (TAGO Inc., Burlingame, CA) at 1,000-fold dilution.
Culture of Lung Fragments Approximately 200 to 300 mg of the lung fragments were incubated with 1 ml of LPS (Escherichia coli 026:B6; Sigma Chemical Co., St. Louis, MO) at 10 p.g/ml, calcium ionophore A23187 (Sigma) at 2.5 p.g/ml, or medium alone in a well of 12-well tissue culture plates (Costar, Cambridge, MA). For IgE receptor triggering, the fragments were at first passively sensitized with 10% of anti-dinitrophenyl (DNP) monoclonal rat IgE in MEM, which was incubated at 56° C for 4 h before passive sensitization in some experiments, for 2 h in tissue culture dishes (35 x 10 rom style; Corning Glass Works, Corning, NY). Anti-DNP monoclonal rat IgE was kindly provided by Creative Products Research Laboratories (Kissei Pharmaceutical Co. Ltd., Matsumoto, Japan) (8). After sensitization, the fragments were washed with MEM sufficiently, and cultured with DNP conjugated to ovalbumin (DNP-OVA) or anti-rat IgE monoclonal mouse IgG (Serotec, Kidlington, UK) as mentioned above. The experiments were performed in duplicate in a 12-well culture plate, and the plates were incubated in the moist chamber gassed with a mixture of 40% O2 and room air at 37° C. After varying incubation periods, the culture supernatants and fragments were harvested. Constituents oftissue culture media were screened for endotoxin contamination using the colorimetric assay of Toxicolor test (Seikagaku Kogyo Co. Ltd., Tokyo, Japan). Contamination of endotoxin was less than 40 pg/ml, which was shown to be unable to stimulate macrophages to produce TNF (18).
Histamine Release from Rat Lung Tissue When the lung fragments were incubated with DNP-OVA at concentrations of 0.01 to 100 p.g/ml after passive sensitization, histamine was released from the fragments dosedependently. Histamine was maximally released after stimulation with 100 p.g/ml of DNP-OVA (12.8 ± 5.1%, n = 3) (Figure 1). When the passive sensitization was performed with medium alone, DNP-OVA at any concentration could not release histamine significantly compared to spontaneously released histamine (data not shown).
Analysis of Histamine Release Approximately 100 mg of the lung fragments with or without passive sensitization were incubated with 0.5 ml of DNPOVA, anti-rat IgE antibody or A23187 at various concentrations in a well of 24-well culture plates (Becton Dickinson) in duplicate. After 1 h, in each well, the histamine content of the culture supernatants (S) and the remaining histamine (L) extracted by boiling the lung fragments for 10 min in phosphate-buffered saline acidified by addition of 12 N HCI (Sigma) to pH 3 to 4 were measured with an automated fluorometric technique (19). The percentage of histamine release in each well was calculated using the following formula:
Cytotoxic Activity in Culture Supernatants of Lung Fragments Culture supernatants of lung fragments were harvested after 1, 2, or 4 h of stimulation (Figure 2). The stimulation with LPS resulted in the release of a high level of cytotoxic activity (279.2 ± 86.4 U/g wet lung, n = 4) after 4 h, and a slight activity (10.2 ± 5.7, n = 3) was detected 4 h after stimulation with A23187, which induced the release of histamine (15.3 ± 6.9). Moreover, the sensitized fragments released the cytotoxic activity 2 and 4 h after stimulation with DNPOVA (44.8 ± 14.6, n = 3 and 106.9 ± 41.5, n = 3, respectively), whereas the incubation of the sensitized fragments
RNA Preparation and Northern Blot Analysis After incubation for various periods, lung fragments were homogenized in 5.2 M guanidine isothiocyanate solution (20). As previously described (8), RNA was extracted and hybridized with mTNF cDNA (provided by Asahi Chemical Industry Co. Ltd.) (21) labeled with (32P]dCTP-a (Amersham International, Buckinghamshire, UK). Statistical Analysis Data were expressed as the mean ± SEM, and statistical significance was assessed using Student's t test.
Results
Ohno, Ohkawara, Yamauchi et al.: IgE Receptor-mediated TNF Production
287
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Figure 1. Histamine release from rat lung tissue with specific antigen. After sensitization, lung fragments were incubated with DNPOVA at concentrations of 0.01 to 100 Itg/ml for 1 h. Experimental results (mean ± SEM, n = 3) were expressed as the percentage of histamine release as described in MATERIAUi AND METHODS.
with medium alone induced the release of only a small level of the cytotoxic activity after 4 h (1.5 ± 1.5, n = 4). On the other hand, the fragments that were passively sensitized with either medium alone or heat-inactivated anti-DNP IgE antibody failed to release cytotoxic activity, even if stimulated with DNP-OVA (data not shown). The incubation with DNPOVA following the passive sensitization with anti-DNP monoclonal IgE has been demonstrated to be able to stimulate IgE receptors on basophils (8). These data suggested that the release of the cytotoxic activity from the lung fragments after stimulation with specific antigen was not due to contamination of endotoxin but to the cross-linkage of IgE receptors. This was confirmed by the evidence that incubation with anti-rat IgE antibody at IOO-fold dilution could induce not only the release of histamine but also that of cytotoxic activity from the lung fragments that were passively sensitized with anti-DNP rat monoclonal IgE antibody, but not from the lung fragments that were passively sensitized with medium alone (data not shown). Neutralization of the Cytotoxic Activity by Anti-mTNF Specific Antibody Anti-mTNF antibody abolished the cytotoxic activity in the culture medium of the lung fragments stimulated with LPS as well as that of recombinant mTNF on L929 cells. In addition, either the cytotoxic activity from the sensitized lung fragments stimulated with DNP-OVA or that from the fragments stimulated with A23187 was completely neutralized after the incubation with anti-mTNF antibody (Table 1). These data suggested that these activities obtained from lung
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Time Ch) Figure 2. Kinetics of cytotoxic activity from rat lung tissue. Lung fragments were incubated with A23187 at 5 Itg/ml (triangles), or DNP-OVA at 100 Itg/ml (circles), or medium alone (crosses) following passive sensitization. After 1, 2, and 4 h, the culture supernatants were assayed for cytotoxic activity and expressed as units per gram of wet lung (mean ± SEM, n = 3 to 4). After stimulation with LPS at 10 Itg/ml for 4 h, 279.2 ± 86.4 (n = 4) of cytotoxic activity was obtained from lung fragments.
fragments had the same immunoreactivity to anti-mTNFspecific serum as mTNF. TNF Gene Expression in Lung Fragments To evaluate TNF gene expression, we hybridized mRNA extracted from rat lung fragments with mTNF eDNA probe (Figure 3). The probe hybridized with RNA from the lung fragments stimulated with LPS. In addition, the hybridized bands with the probe were observed as early as 2 h after IgE receptor triggering at the same position as that after stimula-
TABLE 1
Neutralization of cytotoxic activity by anti-mTNF rabbit serum* % Residual Cytotoxicity after Treatment with: Sample
Lung tissue +LPS +DNP-OVA +A23187 mTNF
Normal Serum
87.3 93.3 76.4 98.0
± ± ± ±
4.4 2.5 8.6 0.9
Anti-mTNF
0.0 0.0 0.3 0.8
± ± ± ±
0.0 0.0 0.3 0.8
* Cultured supernatants of lung fragments and recombinant mTNF were assayed for cytotoxicity after incubation with anti-mTNF rabbit serum as described in MATERIALS AND METHODS. Residual cytotoxicity was calculated according to the following formula: (cytotoxicity with treatment/cytotoxicity without treatment) x 100. Data are expressed as mean ± SEM (n = 4).
288
AMERICAN JOURNAL OF RESPIRATORY CELL AND MOLECULAR BIOLOGY VOL. 3 1990
Medium
o
2
4
+DNP-OVA
8h 2
4
8h
+LPS
2h
285185-
Figure 3. TNF gene expression in rat lung tissue. RNA was extracted at indicated time points after stimulation with medium, DNP-OVA (100 JLg/ml) following passive sensitization, or LPS (10 JLg/ml), and loaded at 20 JLg to each lane. The nitrocellulose filter was hybridized with 32P-labeled mTNF cDNA probe. The migration positions of28S and 18S ribosomal RNA were indicated to the left. An equal amount of RNA in each lane was confirmed with the assessment of 28S and 18S ribosomal RNA stained with acrigine orange after electrophoresis.
tion with LPS, while not after medium alone. An equal amount of RNA in each lane was confirmed with the assessment of 28S and 18S ribosomal RNA stained with acrigine orange after electrophoresis.
Discussion We have demonstrated that the incubation with a specific antigen or anti-IgE antibody following passive sensitization with monoclonal IgE antibody induced not only histamine release but also the gene expression and production of TNF in cultured lung tissue. The contamination of endotoxin in the reagents used in this study was less than 40 pg/ml, which was unable to induce detectable TNF production from macrophages (18). Either specific antigen, anti-IgE antibody, or IgE antibody failed to induce the release of both histamine and cytotoxic activity from the lung fragments when used solely. The passive sensitization with heat-inactivated antiDNP IgE antibody could not induce the release of cytotoxic activity after stimulation with DNP-OVA. These results suggested that IgE receptor-mediated stimulation induced the release of cytotoxic activity from lung tissue. Several laboratories have provided evidence of TNF-like factors in mast cells (5-7). In rat basophilic leukemia cells, we previously showed that the gene expression and production of TNF was induced in response to the cross-linkage ofIgE receptors (8). Recently, using an organ culture of human skin, Klein and colleagues (22) reported that anti-IgE, calcium ionophore, and morphine sulfate induced degranulation of mast cells and expression of an endothelial molecule responsible for leukocyte adhesion, and that the induction of the molecule might be mediated by TNF because anti-human TNF blocked the expression of the molecule. They discussed that mast cells might release a factor with TNF-like properties, by the observation that preincubation with cromolyn sodium, which is an inhibitor of mast cell degranulation, abrogated
the expression of the adherent molecule, though TNF-producing cells were not identified. In our experiments, lung mast cells are the possible cellular source of TNF, and directly express TNF gene transcripts and produce TNF molecules in response to IgE receptor triggering. However, TNF is produced mainly by activated monocytes and macrophages (4), which are also known to express low affinity Fete) receptors (23) and to participate as effector cells in the pathology of allergic reactions through the release of leukotrienes (24), prostaglandins (25), platelet-activating factor (26), and IL-l (27) through IgE receptor triggering. Therefore, it is possible to suggest that macrophages in lung tissue directly release TNF through IgE-mediated stimulation. In addition, there are various types of stromal cells in normal lung tissue such as endothelial cells, vascular smooth muscle cells, epithelial cells, and fibroblasts. Cultured human vascular smooth muscle cells express TNF gene and release TNF protein after stimulation with LPS (28), and TNF mRNA was shown to localize in murine intestinal epithelium (29). These observations raise the possibility that the indirect stimulation of stromal cells through activation of mast cells or macrophages by IgE receptor triggering results in the gene expression and production of TNF in these cells. A23187, which is known to be able to stimulate macrophages to produce TNF (30) and mast cells/basophils to release histamine (31), was shown to induce gene expression and production of TNF in rat basophilic leukemia cells (8) and to activate endothelial cells through the induction of TNF in human skin culture (22). However, we could not determine which cells, macrophages, mast cells, or other types of cells, released TNF after stimulation with A23187. IgE receptor triggering by specific antigen followed by mast cell degranulation play a central role in the pathogenesis of bronchial asthma (1), and, in whole lung tissue studies, it has been observed that chemical mediators such as histamine and leukotrienes were released after antigen challenge (32, 33). However, we demonstrated not only histamine release but also the upregulation of TNF gene and the release of a factor immunologically related to TNF from lung tissue after IgE receptor triggering. Our data represent the first description of cytokine production by whole lung tissue with IgE receptor triggering. On the other hand, it has become clear that TNF contributes indirectly to the recruitment and activation of inflammatory cells such as monocytes, neutrophils, and eosinophils, suggesting that they might attribute to the pathogenesis of late response of bronchial asthma after allergen challenge (34). Therefore, our results may imply the important role ofTNF in the inflammation of bronchial asthma after antigen challenge. Acknowledgments: The writers thank Mrs. Yuko Satoh for technical assistance. This work was supported in part by a Grant-in-Aid for Scientific Research from the Ministry of Education, Science and Culture (Japan).
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