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Lymphocyte Transformation Test with Nickel in Hard Metal Asthma: Another Sensitizing Component of Hard Metal

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Lymphocyte Transformation Test with Nickel in Hard Metal Asthma: Another Sensitizing Component of Hard Metal Industrial Health, 1991, 29, 153-160 153 Lymphocyte Transformation Test with Nickel in Hard Metal Asthma: Another Sensitizing Component of Hard Metal Yukinori KUSAKA*1), Yumiko NAKANO2), Taro SHIRAKAWA3), Naoki FUJIMURA4), Mikio KATO4) and Shinichiro HEKI4) 1) Department of Environmental Health, Jichi Medical School, Minamikawachi-machi, Tochigi-ken 329-04, Japan, 2) Division of Industrial Health, Osaka Prefectural Institute of Public Health, Higashinari-ku, Osaka 537, Japan 3) Department of Hygiene and Preventive Medicine, School of Medicine, Osaka University, Suita City 565, Japan 4) Department of Respiratory Diseases, Takatsuki Red Cross Hospital, Takatsuki City 569, Japan (Received June 28, 1991 and in revised form September 11, 1991) Abstract : To determine cell-mediated immunity to nickel, another matrix in hard metal besides cobalt, lymphocyte transformation tests (LTT) with nickel were carried out in seven hard metal asthma patients all of who had reacted to cobalt chloride in the bronchial provocation tests (BPT). Immunoallergic studies prior to the present study revealed that threee of the seven generated a simultaneous positive reaction in the BPT with nickel and the allergosorbent test with nickel-conjugated human serum albumin (Ni-HSA). A stimula- tion index in LTT indicating a positive response was defined on the basis of results from the studies in the controls. Data revealed that two of the three who showed a combination of positive bronchial and immunological reactions with nickel had a positive LTT with nickel. In the other five, peripheral lymphocytes did not proliferate in response to nickel. Thus it is suggested that cell-mediated immunity to nickel as well as cobalt is implicated in some cases associated with hard metal asthma. Key Words : Asthma-Hard Metal-Cobalt-Nickel-Hypersensitivity-Lymphocyte trans- formation test INTRODUCTION Hard metal asthma, an occupational asthma related to exposure to hard metal, has been shown to develop following inhalation of cobalt salt1-6). Moreover, we have demonstrated that humoral and cellular hypersensitivity to cobalt is im- plicated in onset of some cases associated with hard metal asthma6-8). * To whom correspondence should be addressed . 154 Yukinori KUSAKA, et al. Nickel belongs to the same metallic group (VII) as cobalt. Occupational exposure to nickel salt has been known to cause asthma by allergic process in metal plating workers9-12). Metallic nickel has been used as matrix in hard metal depending on the desired properties of final products5, 13,14) Excess amount of nickel have been identified in the lungs and blood of a patient with hard metal lung disease15)and contact dermatitis from sensitization to both cobalt and nickel was prevalent in the Swedish hard metal industry16-18). We previously described that cases with hard metal asthma mediated through type I hypersensitivity to cobalt also had a positive bronchial provocation test (BPT) with nickel and a positive radioallergosorbent test (RAST) with nickel-conjugated human serum albumin (Ni-HSA)19). Hence it is of interest to assess cell-mediated immunity to nickel among these cases. We report here evidence of cell-mediated immunity to nickel indicated by positive lymphocyte transformation tests (LTT) to nickel in some hard metal asthmatics. SUBJECTS AND METHODS Patients In our previous study8), eight patients asscoiated with hard metal asthma were studied with respect to cell-mediated immunity to cobalt by using LTT. Seven of the eight were subjected to the present study because the rest one did not undergo immunoallergic tests with nickel. For comparison, the initials of the cases used in the text and tables were the same as those appearing in the authors' previous paper on cobalt sensitization8). Controls As controls, we chose four different groups of individuals : (1) 6 healthy non-atopic non-exposed males; (2) 7 healthy hard metal-exposed workers from the same industry who were matched with 7 asthmatic subjects for age, type of work and exposure duration ; (3) 2 atopic but non-asthmatic hard metal workers ; (4) 2 hard metal workers with atopic allergic asthma of non-occupational origin confirmed by negative BPT with cobalt and/or a lack of relationship between attacks and exposure to hard metal dust5). All 17 controls were medically checked by the authors. The blood and skin tests revealed nothing abnormal except in the group 4. Antigens and mitogen ' Nickel sulphate was used as an antigen . NiSO4 • 6H20 of super grade (Wako Chem., Osaka, Japan) was dissolved in physiological saline and applied to the cultures at final concentrations of 1.4, 6.9 and 35 ƒÊg/ml as NiSO4 (Ni1, Ni2, Ni3)24-26). As Ni, the concentrations were 8.9 •~ 10 6M, 4.5 •~ 10-5M and 2.2 X 10-4M, respectively. A polyclonal mitogen stimulation to concanavalin A (Con A, LYMPHOCYTE PROLIFERATION TO NICKEL 155 Sigma) at a concentration of 1 ƒÊg/ml was also performed. Lymphocyte transformation test Peripheral venous blood was drawn from patients and controls. Lymphocytes were separated from heparinized blood by the Ficoll-Hypaque density sedimenta- tion (Pharmacia Fine Chemicals, Sweden), washed three times in Hank's bal- anced salt solution (HBSS, Nissui Co., Tokyo, Japan) and suspended at a concentration of 5•~105cells/m1 in RPMI 1640 medium (Nissui) containing 5% fetal calf serum (Gibco, Grand Island, NY), 1001U/m1 penicillin, 100 ƒÊg/ml streptomycin and 300 ƒÊg/ml L-glutamine. Two hundred ml aliquots of the cell suspensions were cultured in 96-Well flat-bottomed microtitre plates (Nunc, Den- mark). After adding antigens or Con A, the cultures were incubated at 370•Ž in 5% CO2 atmosphere for 72 hours. The rate of cellular DNA synthesis was measured by uptake of 3H—thymidine added 24 hours before cell harvest. Tripli- cate or quadruplicate cultures were prepared for each sample. The results were expressed as a stimulation index (SI) calculated by the following formula : SI = mean count per minute of stimulated culturers/mean count per minute of control cultures. Statistical analysis Differences in the mean values of SI between the controls and the patients were examined by the t-test. A p value of less than 0.05 was considered significant. A normal range of the SI for each antigen was sought. Since the sample number of the controls was small, the normal range was calculated using the t-distribution as follows : Results exceeding this range were considered to indicate significantly positive proliferation of lymphocytes with nickel antigen. RESULTS Lymphocyte transformation test in controls The SI of lymphocyte proliferation with antigens in the controls are shown in Table 1. Since a normal range of SI calculated in the controls was less than two for each antigen, we defined an SI of > 2.0 as indicating a positive lymphocyte transformation test. None of the controls showed a SI of more than two. 156 Yukinori KUSAKA, et al. Table 1. Lymphocyte transfomation (SI) in response to nickel and concanavalin A (Con A) in the controls. Table 2. Lymphocyte transformation reactions (SI) to nickel and Con A in the hard metal asthmatic patients. LYMPHOCYTE PROLIFERATION TO NICKEL 157 Lymphocyte transformation test in hard metal asthmatics Table 2 shows that two cases (A, F) had positive LTT with nickel : lympho- cytes of case A proliferated in response to nickel sulphate of a concentration of 35 ƒÊg/ml , case F's lymphocytes reacted to 1.4 and 6.9 ƒÊg/ml nickel sulphate. Differences in mean SI between the cases and the controls were not significant for each antigen. Mitogenesis with Con A did not differ significantly between the patients and the controls. DISCUSSION The LTT is a useful tool in detecting cell-mediated hypersensitivity to metals such as nickel and beryllium which cause skin or pulmonary lesions20-23). A stimulation index of two or more has been commonly considered to indicate positive sensitization to the metals21,23, 24). Our criteria developed in the current study is in agreement with this. A lack of sensitivity of the LTT which may lead to false negatives has been a matter of debate24). A finding that case B with both positive PT and IgE for nickel did not show a positive LTT might be an example of this insensitivity of the LTT. Concentrations of nickel which induced positive lymphocyte transformations differed among individuals and any dose dependency of the LTT with metal concentration was not seen. Interpretation of these observations is speculative. Con A, a mitogen, was also applied to the test to assess lymphocytes for their ability to proliferate. Enormous differences in mitogenesis with Con A were seen among both the controls and the patients and the mitogenesis did not ditinguish between the patients and the controls. It has been reported in contact dermatitis which is a type IV allergy that lymphocytes proliferating in response to nickel have the phenotype of helper T cell25-26). Again in allergic asthma (type I hypersensitivity), peripheral blood lymphocytes bearing the surface marker for helper T cell have been described to proliferate on stimulation with antigens27-28).Whether it means an involvement of the cell-mediated immunity has not been stated. Of concern is to clarify T cell functions and subsets in metal-induced hypersensitivity. Prior to the present study, the seven patients underwent BPT, PT and RAST with cobalt and nickel, the methods and criteria for which were described previously6-8). Table 3 summarizes the results in the seven patients for those tests in combination with the present study results. It was notable that not all patients having cobalt-provocative asthma produced positive reactions to nickel in the BPT with nickel. As shown in Table 3, patients who reacted to cobalt in the RAST or PT also reacted to nickel in these tests and the patient (Case F) showed a positive response to both cobalt and nickel in the LTT.
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