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Tumour necrosis factor<z (TNF-cz)was identified as an anti-tumour cytokine derived from macrophages (M). So far, the sources of TNF-Materials and Methods 0t include M(, activated T cells, 2 and mast cells. 3'4 TNF-0t has been recognized as having Subjects: We studied 27 patients (15 males and various biological activities relevant to inflamma-12 females, ranging in age from 21 to 32 years) tion, such as expressing adhesion molecules on and eight normal volunteers (four males and endothelium, 5 being chemoattractant, 6 and four females, ranging from 23 to 31 years). enhancing microvascular permeability] Recently, Patients were diagnosed with perennial nasal an increase of TNF-0t in bronchoalveoral lavage allergy according to the following criteria: (1) fluids has been reported and recognized as an perennially persistent and recurrent nasal syrupimportant mediator in bronchial asthma. 8 toms consisting of sneezing attacks, watery nasal However, there have not been sufficient studies discharge and nasal obstruction; (2) positive to clarify the role of TNF-0t in nasal allergy. It eosinophilia in nasal smear test; and (3) house has been reported that T cells elaborate inter-dust-positive intradermal reaction and/or IgE feron-7 (IFN-7) to activate M. 9 Furthermore, antibody against house dust mite, Dermatopbarecently, we elucidated the accumulation of acti-goides farine in the serum. All patients and vated helper T cells in the nasal mucosa of normal subjects gave informed consent. None of allergy patients by measuring soluble interleukin-the patients or controls had other inflammatory 2 receptor (sIL-2R). These reports further diseases and none received any medication suggest that T cells may induce TNF-z elaboraprior to nasal lavage collection. For mRNA analytion by M and mast cells in allergic nasal sis, the nasal mucosa obtained at surgery from mucosa through the cytokine network following four patients with house dust mite allergy was allergen exposure, used. To investigate TNF<z in nasal allergy in the present study, we measured TNF-z in the nasal Assessment of clinical severity of nasal allergy. secretions of allergic patients. We also investi-Nasal symptoms scored in a symptom diary were gated TNF-z mRNA and IFN-7 mRNA in the assessed and the clinical severity was determined nasal mucosa using reverse transcription polyaccording to Okuda's criterion. 1 Frequency of merase chain reaction (RT-PCR) after allergen sneezing attacks, nose blowing and patient-asseschallenge in vitro. Furthermore, to investigate sed grade of nasal obstruction were scored, and the relationship between TNF-a and eosinophils, symptom grades were determined as mild, rood-erate or severe. When one or more of these 2001.tl of TNF-cz conjugate was added after symptoms were severe, overall clinical severity washing three times, then incubated for 1 h at was considered severe. When the symptoms were room temperature. Then 200 tl of substrate solumoderate or mild, overall severity was considered tion was added after washing three times, and moderate. When all of three symptoms were incubated for 2 min at room temperature, folmild, overall severity was considered mild.
lowed by adding 50tl of stop solution. The absorbance was measured at 450nm using an Nasal smear tes Nasal smears were obtained automatic spectrophotometer (Titerteck Multiprior to nasal lavage, and cytologically investi-scan (R) PLUS MKII, Flow Laboratories Inc., CA, gated using Hansel's staining procedure 2 and USA). The absorbance of samples was measured nasal cytograms were graded according to a by the spectrophotometer after calibrating the semi-quantitative scale as reported previously. 3 equipment to 0 using the substrate blank. TNF-0t concentration in the nasal secretion was calcu-Preparation for measurement of TNF<z, and lated according to the above equation. The ECP: Nasal lavage fluids were obtained by minimal detectable dose using a standard curve washing with 20ml of 0.9% saline solution conwas 4.4 pg/ml. taining I mM LiC1, pre-warmed to 37C, using a 30ml plastic syringe with minimal stimulation of Measurement of ECP: The nasal lavage fluids the nasal mucosa. The total volume of recovered from 18 allergic patients and from eight normal lavage fluid was measured and the nasal lavage subjects were radioimmunoassayed (Pharmacia fluid was centrifuged twice at 1350 x g for 10 ECP RIA kit(R), Pharmacia Diagnostics AB, min at 4C. Sputolysin (R) (Behring Diagnostics La Uppsala, Sweden) to determine ECP concentra-Jolla, CA, USA)was mixed with nasal lavage fluid tions as described elsewhere. 5 The minimal at a volume ratio of 0.4/9.0, and immediately detectable ECP was 2 pg/ml.
shaken for 1 min, and then 5.5% aprotinin (Sigma, MO, USA) was added at a volume ratio Reverse transcription polymerase chain reaction: of 0.1/9.4, followed by immediate mixing for 1 min at room temperature. The fluid was allowed Preparation of the nasal mucosa for RT-PCR. to stand for 30 minutes at room temperature, Mucosal specimens were obtained from the then centrifuged twice at 1350 x g for 10 min at inferior turbinates of allergic patients at times of 4C and stored at -80C until TNF-cz and ECP surgery. The specimens were cut into were assayed. 4mm x 4mm pieces, and incubated with or The lithium (Li) concentration in the nasal without extract of Dermatophagoidesfarine at a lavage was measured by atomic emission specconcentration of 1 btg/ml for 15 min in RPMI trophotometry to calculate TNF-0t and ECP con-I640, and then incubated at 37C for 3 h. centrations in the nasal secretions. Li was used as an exogenous marker of nasal secretion allowing Extraction of RNA. RNA was extracted from the calculations to be made from small amounts of mucosal specimens using RNeasy TM kit (QIAGEN, nasal secretion. 4 The TNF<z concentration (TNF-CA, USA). After treatment with DNase (Promega, x) was calculated by the following equation: WI, USA) at a ratio of 1 unit/tg RNA, to clean up the RNA, the RNA product was treated with the
where TNF-CZn denotes the TNF-cz concentration Preparation of cDNA. The RNA was incubated at of the sample; Lio, the Li concentration of the 65C for 10 min. One tg of the RNA, 2 !1 of 10 0.9% NaC1 for lavage; and Lin, the Li concentra- x PCR Buffer II, 2 l.tl of 25 mM MgCl2 (Perkintion of the sample. The ECP concentrations were Elmer, NJ, USA), 411 of 2.5 mM d-NTPs (dATP, calculated by the same equation using ECP dCTP, dGTP and dTTP; Pharrnacia, NJ, USA), l btl instead of TNF-cz. of 20 U/l.tl RNasin (Wako, Osaka, Japan), 1 !,1 of 100 pM random primer, 1 !1 of 200 unit/l.tl of Mo-Measurement of TNF<z by enzyme-linked immu-MLVReverse Transcriptase (Gibco BRL, NY, USA) nosorbent assay: Enzyme-linked immunosorbent were mixed and adjusted to 20 I1 in total volume Tt assay (Quantikine R&D Systems, Inc. MN, by adding DEPC-water. The reaction mixture USA) was used to detect TNF<z in nasal lavage was allowed to stand at room temperature for 10 fluids. The assay was performed in duplicate, min, then incubated at 42C for 60 min, and at Briefly, 200 btl of standard or sample was added 95C for 5 min in a water bath. The obtained to 50 tl of the diluent and incubated at 37C for reverse transcription solution (cDNA) was stored 2h in a fiat-bottomed 96-well microplate. Then at-20C.  tion of TNF-t of patients with mild nasal allergy showed a significantly higher level than that of normal subjects (p < 0.05). There was no correlation between TNF-cz concentration and clinical severity (Fig. 1). ECP in nasal secretions: The mean (+_ S.E.) value of ECP concentration in the nasal secretions of allergic patients was 270.1 Jr 68.51.tg/1 (n--19), and it was significantly higher than that of normal subjects (less than the minimal detection level of the assay, n=8, p<0.01).
However, there was no correlation between ECP and TNF-cz concentrations in the same nasal secretions (r 0.089, Fig. 2).  Table 2. The ratios of the OD of mRNAs/the OD of -actin mRNA in the nasal mucosa. The ratios of the OD of mRNAs of TNFand IFN-7/that of -actin showed significant differences from the controls 3 h after allergen challenge, although the base line values of neither mRNA showed any significant differences between the groups with and without allergen challenge (n=4). There were decreases in the OD ratios of IFN-? and controls after allergen challenge because of increased OD of -actin mRNA. were significantly higher than those without allergen challenge, although there was no significant difference between the base line levels of either mRNA with allergen challenge and those of the controls (n 4, Table 2, Fig. 3).

Discussion
Specific reaction of the allergen with IgE antibodies bound to mast cells causes immediate and late nasal responses. The mast cells release chemical mediators and also elaborate cytokines such as IL-3,16 IL-4, IL-5, IL-6, GM-CSF 17 and TNFoz.
These cytokines may take part in the network and modulate allergic inflammation of the nasal mucosa.
Eosinophil accumulation in the nasal mucosa is a hallmark of nasal allergy, and nonspecific hyperresponsiveness of the nasal mucosa is an important clinical feature of nasal allergy. Eosinophils predominantly release lipid mediators such as leukotriene C4 (EWe4) 19 and platelet activating factor (PAF) 2 as well as cytotoxic granule proteins such as major basic protein, ECP, eosinophil peroxidase and eosinophil derived neurotoxin. 21 Recent studies have suggested that eosinophils play an important role in the mechanism of mucosal nonspecific hyperresponsiveness. Therefore, eosinophil recruitment in the nasal mucosa is crucial in the pathogenesis of nasal allergy.
In the present study, TNF-cz was detected in nasal lavage fluid from patients with perennial nasal allergy. Bachert et a/. 22 reported that TNF-z increased in nasal lavage during immediate nasal response. Some previous studies have reported that this cytokine was detected or not detected in nasal lavages from nasal allergy patients. 23 '24 Inconsistent results are presumably due to rapid metabolism of this cytokine in the nasal lavage fluids and/or the small amount present, so that the level was undetectable in some cases by ELISA. It is also surmised that TNF<z might be converted rapidly during transfer into the nasal secretions from the nasal mucosa following the allergic response. We used a protease inhibitor, aprotinin, in the nasal lavage fluid and this possibly resulted in a successful detection of TNF-z in the nasal lavage in the present study, although the level of TNF-cz in nasal secretions did not show any relation either to clinical severity, or to the ECP level in nasal secretions. On the other hand, the ECP level in nasal secretions showed a positive relationship to clinical severity in our previous study. 5 Recently, human mast cells were found to contain preformed TNF-cz which is capable of release by immunological activation. 3'4'23 These data suggest that TNF-cz may be released and play a role in immediate nasal response.
TNF-cz was reported to be derived from acti-vated M. To date, it has been reported that not only M but also mast cells and T cells generate TNF<z, which has various biological activities such as promotion of: (1) oxygen radical production of eosinophils and neutroohils; v (2) mast cell and eosinophil cytotoxicity; 2'26 (3) microvascular permeability; v (4) leukotriene B4 (LTB4) and PAF generation from eosinophils and neutrophils; 2v'2. and (5) eosinophil and neutrophil adhesion to the endothelium and also airway epithelium by expression of adhesion molecules. 5'29 Furthermore, it is a chemoattractant for neutrophils and monocytes. These biological activities lead to a hypothesis that TNF-a may amplify allergic inflammation resulting in mucosal hyperresponsiveness. Reports that TNF-a induces airway hyperresponsiveness in experimental animals'--support this hypothesis.
It has been reported that TNF<z induces expression of adhesion molecules such as ICAM-1, VCAM-1 and ELAM-1 on vascular endothelial cells, 29'32'33'34 which may result in the adhesion of inflammatory cells to the endothelium and transmigration into the lesion. It is well known that LTB 4 and PAF are potent chemoattractants for eosinophils 5' despite the fact that these lipid mediators do not specifically act on eosinophils like IL-5. v Therefore, TNF-z might directly or indirectly be involved in late nasal response with infiltration of eosinophils and/or other inflammatory cells in allergic nasal mucosa by promoting the generation of these lipid mediators of inflammatory cells. Thus, to clarifi7 whether TNF<z is generated or not after allergen challenge, the TNF<z mRNA in the allergic nasal mucosa was examined using specific RT-PCR because of presumable small amount of TNF-z in the culture supernatant.
Specific RT-PCR revealed TNF<z mRNA in the allergic nasal mucosa before and after allergen challenge in vitro in the present study. TNF<z mRNA specifically increased in the allergic nasal mucosa after allergen challenge. These data suggest that allergen exposure positively induces TNF<z generation in allergic nasal mucosa resulting in inflammatory cell recruitment. This hypothesis is supported by positive hybridization signals for TNF-0t mRNA and a significant increase in the number of TNF-0t mRNA-positive cells in the nasal mucosa of allergic patients after nasal allergen challenge.
The present study also revealed that TNF-z concentrations in nasal secretions did not correlate with either clinical severity or concentrations of ECP in the nasal secretions obtained from allergic patients. As previously described, TNF-z has various biological activities relevant to accumulation and activation of inflammatory cells involved in allergic reactions. Consequently, these negative correlations suggest that TNF<z may indirectly play a role in causing clinical symptoms, and other factors may collaborate with TNF<z in eosinophil activation. In other words, TNF<z may play a central role in the cytokine network in the nasal allergy by regulating inflammatory cells such as lymphocytes, macrophages, mast cells and eosinophils.
Our previous studies suggested activation of helper T cells in nasal allergy by elevation of slL-2R level in nasal secretions and also cluster formation of CD25 + cells in the allergic nasal mucosa. Activated T cells are capable of generating IFN-7, which is a M activating factor that promotes TNF-z and IL-1 production of M(. 39 IFN-7 also promotes TNF action by increasing TNF receptor expression on sensitive cells. 4 In the present study, IFN-7 mRNA and TNF<z mRNA were determined in the same allergic nasal mucosa following allergen exposure. This result suggests a possibility that T cell-derived IFN-7 may take part in up-regulation of TNF<z amplifying sequential allergic events in the allergic nasal mucosa collaborating with TNF<z derived from other inflammatory cells such as T cells and mast cells. Details of cell-to-cell interaction through the cytokine network relevant to TNF-z remain to be clarified in nasal allergy.