Organic dust-induced lung injury and repair: Bi-directional regulation by TNFα and IL-10.

Exposure to organic dust increases chronic airway inflammatory disorders. Effective treatment strategies are lacking. It has been reported that hog barn dust extracts (HDE) induce TNFα through protein kinase C (PKC) activation and that lung inflammation is enhanced in scavenger receptor A (SRA/CD204) knockout (KO) mice following HDE. Because interleukin (IL)-10 production can limit excessive inflammation, it was hypothesized here that HDE-induced IL-10 would require CD204 to effect inflammatory responses. C57BL/6 wild-type (WT), SRA KO, and IL-10 KO mice were intranasally challenged daily for 8 days with HDE and subsequently rested for 3 days with/without recombinant IL-10 (rIL-10) treatment. Primary peritoneal macrophages (PM) and murine alveolar macrophages (MH-S cells) were treated in vitro with HDE, SRA ligand (fucoidan), rIL-10, and/or PKC isoform inhibitors. HDE induced in vivo lung IL-10 in WT, but not SRA KO mice, and similar trends were demonstrated in isolated PM from same treated mice. Lung lymphocyte aggregates and neutrophils were elevated in in vivo HDE-treated SRA and IL-10 KO mice after a 3-d recovery, and treatment during recovery with rIL-10 abrogated these responses. In vitro rIL-10 treatment reduced HDE-stimulated TNFα release in MH-S and WT PM. In SRA KO macrophages, there was reduced IL-10 and PKC zeta (ζ) activity and increased TNFα following in vitro HDE stimulation. Similarly, blocking SRA (24 hr fucoidan pre-treatment) resulted in enhanced HDE-stimulated macrophage TNFα and decreased IL-10 and PKCζ activation. PKCζ inhibitors blocked HDE-stimulated IL-10, but not TNFα. Collectively, HDE stimulates IL-10 by an SRA- and PKCζ-dependent mechanism to regulate TNFα. Enhancing resolution of dust-mediated lung inflammation through targeting IL-10 and/or SRA may represent new approaches to therapeutic interventions.

Farmer's lung in a case after bullectomy.

We present a case of farmer's lung (FL) with the primary presenting feature of a large bulla in the lung. A 70-year-old nonsmoking woman with dyspnea on exercise was referred for surgical resection of a large bulla in the lung. The postoperative evaluation of the lung tissue revealed a follicular lymphocytic alveolitis and loosely formed granulomas suspicious for hypersensitivity pneumonitis (HP). The patient had worked in farming since her youth. Dyspnea on exercise was the only symptom, but it was related to the large bulla. No other radiologic features of HP were shown in a high-resolution CT of the lung. Specific IgG antibodies against typical antigens of FL were detected, bronchoalveolar lavage demonstrated no lymphocytic alveolitis but an inhalative challenge with own hay was positive. A diagnosis of chronic FL was made. Despite lung emphysema being a possible reaction in FL, giant bullae as primary and single manifestation of this disease have not been reported before.

Bronchoalveolar lavage in hypersensitivity pneumonitis: a series of 139 patients.

Hypersensitivity pneumonitis (HP) is characterized by a lymphocytic alveolitis, classically delineated by an increase of CD8+ lymphocytes, with an inversion of the CD4+/CD8+ ratio. The aim of this study is both to describe the yield and cell bronchoalveolar lavage (BAL) profile and to revisit the assumption of low BAL CD4/CD8 ratio in the diagnosis of HP. A multicentric study was conducted on 139 patients who fulfilled the standardized diagnostic criteria of HP, mainly affected by farmer's lung. Mean total cell count in BAL fluid was 594 ± 401.10(3) cells /ml. Prominent absolute lymphocytic alveolitis, moderate neutrophilia, and mild eosinophilia and mastocytosis were found. Mean CD4/CD8 ratio was 3.8 ± 6.1 (median 2.1). Thirty four percent of the patients showed lymphocytic CD8 alveolitis (ratio < 1). The CD4/CD8 ratio was not different between forms, etiologies of HP, and time elapsed since last antigen exposure, but was higher in women (p=0.02). BAL in HP shows high total cell and lymphocyte counts, moderate neutrophilia, and mild eosinophilia and mastocytosis. An absence of low CD4/CD8 ratio should not at all exclude diagnosis.

Farmer's lung-induced hypersensitivity pneumonitis complicated by shock.

Hypersensitivity pneumonitis is an immunologic reaction to an inhaled antigen, with a wide spectrum of clinical presentations. The most common manifestations are fever, cough, and dyspnea. We describe a case of hypersensitivity pneumonitis with marked alveolar lymphocytosis; the patient presented with respiratory failure and shock requiring mechanical ventilation and vasopressive agents. We hypothesized that immune mediators implicated in the pathogenesis of hypersensitivity pneumonitis were responsible for the transient shock observed in this patient.

New evidence of the involvement of Lichtheimia corymbifera in farmer's lung disease.

Farmer's lung disease (FLD) is a form of hypersensitivity pneumonitis resulting from recurrent exposure to moldy plant materials. We investigated and compared the initial response of respiratory epithelium after exposure to extracts of Sacharopolyspora rectivirgula, Lichtheimia corymbifera (formerly Absidia corymbifera), Eurotium amstelodami and Wallemia sebi. The two criteria for selection of these species were their high prevalence in the hay handled by FLD patients and the presence of high levels of specific precipitins to these molds in FLD patients’ sera. Hydrosoluble extracts were prepared from spores and hyphae grown in culture under optimal conditions for each of the four species. Confluent A549 cells were inoculated with one of the four calibrated soluble extracts. Two mediators, one inflammatory (Interleukin (IL)-8) and one allergic (IL-13), were quantified using real-time PCR and ELISA assay, after four exposure periods (30 min, 2 h, 4 h and 8 h). S. rectivirgula and L. corymbifera extracts were the only ones which induced a marked upregulation of IL-8, as shown by both real-time PCR and ELISA assay 8 h after the initial contact. This study adds to the growing body of evidence that L. corymbifera should be recognized as an etiologic agent of FLD along with S. rectivirgula.

[Farmer's lung disease caused by straw or hay? Case report]. / Hálmsótt eda heysótt?

A young woman was admitted to Akranes Regional Hospital because of dyspnea, fatigue and fever. She was found to have bilateral pneumonia but etiology was not found. She was treated with antibiotics with good resolution and was discharged after eight days from the hospital. Four weeks later she noticed rapidly progressive dyspnea and was found to be hypoxemic, and to have restrictive spirometry and diffuse interstitial changes on chest radiography. Computerized tomography of the lungs showed diffuse ground glass changes. Transbronchial biopsies from the lungs showed numerous small granulomas. She was treated with prednisolone for a short time with excellent recovery. She stall-fed horses and underneath them was straw containing organic dust. This is important to keep in mind as a differential diagnosis to farmers lung disease that is caused by hay.

Serum KL-6 concentrations in dairy farmers.

STUDY OBJECTIVES: Serum KL-6 (Krebs von den Lungen-6) has been recognized to be a marker for the activity of diffuse interstitial lung diseases. The purpose of the study is to evaluate serum KL-6 measurement as a marker for farmer's lung disease (FLD). DESIGN: A cross-sectional survey of a cohort of dairy farmers. Retrospective measurement of KL-6 stored serum samples from those dairy farmers previously screened for FLD. SETTING: University hospital screening project for FLD within a dairy-farming community in Japan. PARTICIPANTS: Four hundred seventy-two dairy farmers were invited to attend a local clinic. MEASUREMENTS AND RESULTS: We examined serum KL-6 concentrations in 272 farmers. Subjects were classified into three groups: (1) 5 farmers with FLD, (2) 30 farmers with positive serum precipitating antibodies to Saccharopolyspora rectivirgula and/or Thermoactinomyces vulgaris without FLD (Ab(+)), and (3) 237 farmers without these antibodies (Ab(-)). Serum KL-6 concentrations in the FLD group were significantly higher than those in the Ab(+) and the Ab(-) groups (1,263 +/- 288 [SEM], 328 +/- 57, and 207 +/- 6 U/mL, respectively, p < 0.001). Serum KL-6 concentrations in those with FLD were significantly higher than KL-6 concentrations from stored screening samples from the same individual when FLD was not diagnosed (1,263 +/- 288 and 419 +/- 209 U/mL, respectively, p < 0.05). Serum KL-6 concentrations of the Ab(+) group were significantly higher than those of the Ab(-) group (p < 0.001). In the Ab(+) group, farmers with high serum KL-6 concentrations had lower permeability coefficients than farmers with normal serum KL-6 concentrations (p < 0.05). These results may suggest that subclinical FLD can be detected in farmers with high KL-6 concentrations and precipitating antibodies. CONCLUSION: Serum KL-6 concentration can be a useful marker for assessing the activity of FLD and may be able to be used to detect subclinical disease.

IgG antibodies, chronic bronchitis, and pulmonary function values in farmer's lung patients and matched controls.

We measured IgG antibody levels against eight different microbes in farmer's lung (FL) patients an average of 14 years after the first diagnosed episode of FL and in matched controls. The study population consisted of 87 FL patients and 81 control farmers, matched by age, sex, and smoking habits. Clinical studies included the measurement of IgG antibody levels against Absidia corymbifera, Aspergillus umbrosus, A. fumigatus, Humicola grisea, Saccharopolyspora rectivirgula, Penicillium brevicompactum, Rhodotorula glutinis, and Thermoactinomyces vulgaris, in addition to spirometry, pulmonary diffusing capacity (DL(CO)), and the evaluation of chronic bronchitis. Median IgG antibody levels were two or more times higher in FL patients than control farmers against Ab. corymbifera, S. rectivirgula, and T. vulgaris (P<0.001). Against A. fumigatus, H. grisea, and R. glutinis, FL patients also had significantly higher antibody levels. FL patients often had positive antibody titers against several microbes, whereas control farmers usually had a positive titer against one or two microbes. A positive association between IgG antibody levels and chronic bronchitis and DL(CO) was observed in FL patients, but not in control farmers. It is suggested that the high antibody levels noted in FL patients were due not only to high exposure but also to individual sensitivity to environmental microbes.

TNF receptor-deficient mice reveal divergent roles for p55 and p75 in several models of inflammation.

The pleiotropic activities of the potent proinflammatory cytokine TNF are mediated by two structurally related, but functionally distinct, receptors, p55 and p75, that are coexpressed on most cell types. The majority of biologic responses classically attributed to TNF are mediated by p55. In contrast, p75 has been proposed to function as both a TNF antagonist by neutralizing TNF and as a TNF agonist by facilitating the interaction between TNF and p55 at the cell surface. We have examined the roles of p55 and p75 in mediating and modulating the activity of TNF in vivo by generating and examining mice genetically deficient in these receptors. Selective deficits in several host defense and inflammatory responses are observed in mice lacking p55 or both p55 and p75, but not in mice lacking p75. In these models, the activity of p55 is not impaired by the absence of p75, arguing against a physiologic role for p75 as an essential element of p55-mediated signaling. In contrast, exacerbated pulmonary inflammation and dramatically increased endotoxin induced serum TNF levels in mice lacking p75 suggest a dominant role for p75 in suppressing TNF-mediated inflammatory responses. In summary, these data help clarify the biologic roles of p55 and p75 in mediating and modulating the biologic activity of TNF and provide genetic evidence for an antagonistic role of p75 in vivo.

Interferon-gamma is necessary for the expression of hypersensitivity pneumonitis.

Farmers lung disease is a common form of hypersensitivity pneumonitis (HP) and is characterized by inflammation and granuloma formation in the lung. Interferon-gamma is important for the expression of granulomatous diseases caused by infectious agents; however, the role this mediator in regulating expression of the granulomatous response to inhaled antigen is not known. To evaluate this, we compared the response to inhaled antigen of mice that do not express the gene coding for interferon-gamma (GKO) with that of their normal littermates (WT). GKO and WT mice on a BALB/c background were exposed to 150 microg of the thermophilic bacteria Saccharopolyspora rectivirgula or saline alone, for three consecutive days a week, for 3 wk. After exposure to antigen, WT mice developed a marked granulomatous inflammation associated with an increase in lung weight and numbers of cells in bronchoalveolar lavage fluid (BAL). Although GKO mice also exhibited an increase in lung weight and numbers of cells in BAL fluid, they developed minimal inflammation and no granulomas after a similar exposure to antigen. To further evaluate if the lack of a response to antigen in GKO mice was due to lack of IFN-gamma, we replaced this mediator via intraperitoneal injections. When given replacement IFN-gamma, the GKO mice developed granulomatous inflammation in the lung. These studies show that IFN-gamma is essential for the expression of hypersensitivity pneumonitis.

[Chronic obstructive bronchitis in a fodder farming setting]. / La bronchite chronique obstructive (BCO) en milieu agricole fourrager.

Respiratory disease in agricultural workers are dominated both in terms of frequency and severity by chronic obstructive long disease. In the Doubs, in dairy cereal farmers, the prevalence of chronic bronchitis in active farm workers is around 10 per cent, and 6 per cent are non-smokers. It is twice the level of the control population of non-exposed subjects. The frequency is most elevated in the men and increases both with age and with altitude. A longitudinal study of male farm workers aged more than 45 has shown that there is an abnormally rapid loss of forced expired volume in one second (FEV1). Two different studies carried out in the Doubs have revealed epidemiological, respiratory function and immunological arguments in favour of immuno-allergic mechanisms. Notably, obstructive chronic bronchitis (OCB) occurs more frequently in agricultural workers with a previous history of farmer's lung or previous sub-acute delayed symptoms in relation to exposure. On the other hand, there are no close links between OCB and the intensity of antigenic exposure (the total quantity of fodder handled). By contrast, there is a relationship between exposure to thermophillic actinomycetes (antigen of farmer's lung) and OCB. The frequency of serum precipitins is most elevated in farm workers with OCB than in asymptomatic agricultural workers. Finally, non-smokers who are suffering from OCB without evidence of farmer's lung, have a respiratory function profile and also alveolar lavage cell pattern characteristic of extrinsic allergic alveolitis after provocation tests to mouldy hay. These arguments are in favour of immuno-allergic mechanisms in the pathogenesis of chronic bronchitis in farm workers which seems to be well differentiated from chronic bronchitis due to smoking.

Altered immunosuppressive activity of alveolar macrophages in farmer's lung disease.

Since normal alveolar macrophages (AMs) can suppress T-cell proliferation to mitogenic and antigenic stimuli both in vitro and in vivo, we questioned whether an altered AM immunosuppressive activity could account for the alveolar lymphocytosis observed in farmer's lung (FL) and whether granulocyte/macrophage colony-stimulating factor (GM-CSF), a cytokine able to abrogate AM-induced immunosuppression, is involved in the process. The ability of different concentrations of AMs to inhibit lymphocyte proliferation in response to the T-cell-specific mitogen phytohaemagglutin (PHA) after in vitro culture was tested in three groups of subjects: 12 patients with FL; four asymptomatic farmers (AS); and six normal volunteers (N). Release of GM-CSF by AMs was also measured. At all ratios tested, AMs from patients with FL did not suppress the lymphoproliferation but instead had an enhancing effect. In AS, AMs enhanced the proliferation at a lower ratio but inhibited it at high ratios. In N subjects, as described previously, AMs increasingly inhibited the blastogenesis of lymphocytes (L) at increasing ratios of AM:L. In some patients with FL, AMs spontaneously released more GM-CSF than in normal volunteers (206 +/- 84 versus 29 +/- 14 pg.mL-1, respectively). In AS, GM-CSF release was intermediate (74 +/- 36 pg.mL-1). In conclusion, a defect in the ability of alveolar macrophages to suppress the proliferation of lymphocytes in the lung of patients with farmer's lung is a major factor accounting for the development of the observed lymphocytic alveolitis. Granulocyte/macrophage colony-stimulating factor could be one factor which may contribute to this alteration.

Long-term viral enhancement of lung response to Saccharopolyspora rectivirgula.

The current study was done to look at the long-term enhancing effect of a single viral infection on repeated Saccharopolyspora rectivirgula (SR) antigenic challenges in mice and at inflammatory cytokines in this enhancement. Four groups of C57BI/6 mice were studied: Group 1 received intranasal instillations of saline, 3 days per week; Group 2, intranasal instillations of saline plus one intranasal instillation of 80 hemagglutination units (HAU) of Sendai virus after 3 wk of saline; Group 3, instillations of SR, 3 days per week; and Group 4, instillations of SR, 3 days per week, plus one instillation of Sendai virus after 3 wk of SR. Bronchoalveolar lavages (BAL) were performed 15 and 30 wk after the virus inoculation in the appropriate groups. Each time, a two- to threefold increase in BAL cell counts was obtained from virus-infected animals challenged with SR compared with animals that received the SR alone. Animals infected with virus only showed values similar to those of control animals. A higher percentage of large foamy multinucleated cells were found in the BAL from the SR+Sendai group than the SR alone group (7.92 +/- 0.730% compared with 1.8 +/- 0.296%). These cells were not seen in the other groups. BAL levels of TNF-alpha and IL-1 alpha at 15 wk were much higher in SR+Sendai-treated (1,439 +/- 268 and 96 +/- 9 pg/ml, respectively) than SR alone-treated animals (361 +/- 100 and 23 +/- 4 pg/ml). BAL fluid of control animals and Sendai alone animals contained 64 +/- 24 and 65 +/- 15 pg/ml of TNF-alpha, but no IL-1 alpha was detected.(ABSTRACT TRUNCATED AT 250 WORDS)

A study of monokine release and natural killer activity in the bronchoalveolar lavage of subjects with farmer's lung.

We examined the interleukin-1 (IL-1) and tumor necrosis factor alpha (TNF alpha) releasability of alveolar macrophages and the natural killer (NK) cell activity in the bronchoalveolar lavage (BAL) cells of 11 patients with Farmer's lung at different stages of the disease. Although there were some variations in the levels of monokine release, macrophages of patients with acute disease secreted significantly higher spontaneous levels of TNF alpha than did a nonfarming control group (p = 0.0002). Conversely, TNF alpha release stimulated by bacterial lipopolysaccharide (LPS) was similar in patients with acute disease when compared with that in normal control subjects. IL-1 was also spontaneously secreted in significantly greater amounts by patients with acute Farmer's lung than by subjects in a control group (p = 0.0001). However, LPS-induced IL-1 release was significantly diminished in BAL macrophages from patients with acute manifestations of the disease when compared with that in control subjects (p = 0.001). Treating hypersensitivity pneumonitis with corticosteroids or by contact avoidance resulted in very significant decrease in spontaneous and LPS-stimulated IL-1 production by BAL macrophages (p = 0.0001 and p = 0.03, respectively), as well as in a decrease in spontaneous TNF alpha release that was also significant (p = 0.01). In addition, BAL cells of patients in the acute phase had a significant NK cell activity (mean +/- SEM of 18.33 +/- 2.65%). Treatment of these patients resulted in an increase in NK cell activity (mean of 40.17 +/- 7.86%), which was significantly different from values of patients with acute disease (p = 0.037).(ABSTRACT TRUNCATED AT 250 WORDS)

[Case report of Farmer's lung disease in siblings in Kagoshima Prefecture].

Two cases of farmer's lung disease in siblings are reported. A 54-year-old male farmer, who had been engaged in stock work for 20 years, presented to our clinic for the second episode of fever, productive cough and shortness of breath. Chest roentgenogram revealed diffuse micronodular pattern, and mild hypoxemia was recognized on arterial blood gas analysis. Cytology obtained from BALF showed lymphocytosis, with especially increased OKT3, OKT4 positive cells and OKT4/8 ratio. The diagnosis was confirmed by highly positive reaction of precipitins to Thermoactinomyces vulgaris and granulomatous interstitial pneumonitis on histopathological examination. The second case was a 51-year-old female patient, the sister of the first case, who also worked as a stock farmer of another farm for 20 years. She presented with an episode of similar symptoms to the first case, one and a half years after the onset of her brother's symptoms. The findings of roentgenogram, BALF analysis, precipitins to T. Vulgaris and pathology were similar to those of the first case. The finding of high OKT4/8 ratio on BALF analysis in both cases is characteristic of formers lung disease, in contrast to the summer-type hypersensitivity pneumonitis which usually shows low OKT4/8 ratio in BALF. To our knowledge, this is the first report of farmer's lung disease in an area other than the northern part of Japan. Thus we conclude that farmer's lung disease may occur in Japan under any environmental conditions, and that some genetic factor may take part in the onset of this disease.

A role for IgE in extrinsic allergic alveolitis?

Receptors for the Fc portion of immunoglobulin E (IgE; CD23) can be detected on the surface of alveolar macrophages (AM) in extrinsic allergic alveolitis (EAA), using monoclonal antibodies in immunocytology. More than 50% of AM were positive in 16 of the 20 patients reported here, while the remaining 4 had 11-47% positive cells. Staining with anti-IgE antibody can, in addition, demonstrate endogenous IgE bound to the AM. This suggests that IgE might be involved in the process. Since IgE-mediated asthma is associated with bronchoconstriction, we asked whether EAA patients do in fact exhibit an obstructive component. In 3 out of 10 patients we did indeed find clearly increased airway resistance (greater than 30 kPa x s x l-1). These findings are consistent with the observation of immediate bronchoconstriction observed in some patients upon allergen challenge. Since only 1 of the 20 patients studied was a smoker, and since in the literature the majority of reported cases of EAA are in nonsmokers, we speculate that smoking may interfere with immunological processes leading to EAA.

Difference in the phenotypes of bronchoalveolar lavage lymphocytes in patients with summer-type hypersensitivity pneumonitis, farmer's lung, ventilation pneumonitis, and bird fancier's lung: report of a nationwide epidemiologic study in Japan.

We performed a nationwide epidemiologic study of hypersensitivity pneumonitis (HP) in Japan by questionnaire and found that 835 cases of HP were recognized during the 1980s; 74.4% were summer-type HP, 8.1% farmer's lung, 4.3% ventilation pneumonitis, 4.1% bird fancier's lung, 2.3% other types, such as chemical worker's lung, and 6.8% of unknown causative agent. It was found that the CD4/CD8 ratios of bronchoalveolar lavage (BAL) lymphocytes were significantly different with the type of disease. The ratio was 0.6 +/- 0.1 (mean +/- SEM) in summer-type HP (N = 271), 4.4 +/- 0.7 in farmer's lung (N = 22), 1.6 +/- 0.3 in ventilation pneumonitis (N = 19), and 2.0 +/- 0.5 in bird fancier's lung (N = 19). In farmer's lung, the CD4/CD8 ratio in smokers was 6.2 +/- 1.9 (N = 6) in contrast with 3.4 +/- 0.7 for nonsmokers (N = 16) (p less than 0.05). It has been generally considered that the phenotypes of BAL lymphocytes in patients with HP are predominately CD8 cells. Our present results, however, indicate that the phenotypes of BAL lymphocytes vary with the type of HP, probably depending on factors such as causative agent, smoking, or staging of the disease.

Enhanced expression of CD2 antigen on lung T cells.

CD2 molecules not only function in the adhesion of T cells to other cell types but also in the activation of T cells via an alternative pathway. To investigate the possible roles of CD2 molecules in the activation and accumulation of lung T cells, using monoclonal antibodies and a flow cytometer we evaluated CD2 antigen expression on lung and blood lymphocytes in 10 normal subjects, 30 patients with pulmonary sarcoidosis, 7 patients with interstitial pneumonia associated with collagen vascular disease, 5 patients with farmer's lung disease, and 8 patients with Crohn's disease. The mean fluorescence intensity (MFI) of CD2 on lung T cells obtained by bronchoalveolar lavage (BAL) was significantly higher than that on blood T cells in all study groups except the control group. In patients with pulmonary sarcoidosis this enhancement of the expression of CD2 antigen was observed only on CD4+ T cells. Since ligands for CD2 are present on immunocompetent cells of other types in the local pulmonary milieu, these results suggest that increased expression of CD2 molecules could facilitate the communication and interaction of lung T cells with their environment in the lung and thereby possibly contribute to the local accumulation of T cells.

Five-year follow-up of Micropolyspora faeni antibody in smoking and nonsmoking farmers.

To examine factors that influence changes in Micropolyspora faeni (MF) antibody titer in farmer's lung disease (FLD), we followed for 5 yr the prevalence of serum MF antibody and the epidemiologic factors (years on farm, hours in barn, and hay-handling time) among 92 dairy farmers in Hokkaido, Japan. The prevalence of MF antibody among nonsmokers was significantly higher than that among smokers: 27.1% versus 7.7% in 1979, 31.3% versus 2.9% in 1984. There was no remarkable change in overall prevalences of MF antibody between 1979 and 1984 (18.5 and 19.6%, respectively). However, out of 17 seropositive farmers, six (35%) became seronegative, and out of 75 seronegative farmers, seven (9.3%) became seropositive after 5 yr. The nonsmoking farmers who remained seronegative throughout the follow-up period were older and had worked longer on farms than the farmers with seroconversion. These results suggest that in addition to smoking habits, age and exposure time to MF influence the immune response to MF in dairy farmers. Out of 11 farmers who remained seropositive throughout the 5-yr period, two (18.2%) developed FLD. Therefore, continuously positive MF antibody is one of the risk factors in the development of FLD.