CLINICAL AND DIAGNOSTIC LABORATORY IMMUNOLOGY, Mar. 1994, p. 164-171 Vol. 1, No. 2 1071-412X/94/$04.00+0 Copyright (C 1994, American Society for Microbiology Novel In Vitro Method for Identification of Individuals at Risk for Beryllium Hypersensitivity EMMANUEL A. OJO AMAIZE,* MELKON S. AGOPIAN, AND JAMES B. PETER Specialty Laboratories, Inc., Santa Monica, California 90404-3900 Received 17 August 1993/Returned for modification 27 September 1993/Accepted 14 November 1993 Beryllium-specific lymphocytes were generated by in vitro immunization of peripheral blood mononuclear cells (PBMC) from healthy unexposed individuals. Measurement of blastogenic responses of PBMC by [3H]thymidine uptake demonstrated that sensitization of PBMC with beryllium salts followed by stimulation with unrelated salts resulted in a negative response, whereas sensitization and restimulation of PBMC with beryllium salts produced a positive response. Flow cytometric and cell depletion analyses showed that all of the responding cells were CD4+ T cells. The in vitro immunization system was used to screen 52 human subjects for susceptibility to beryllium sensitization in vitro. The results show that of the 52 healthy unexposed subjects tested, only 1 (2%) was highly responsive, 4 subjects (8%) were moderately responsive, 20 subjects (39%) were low-level responders, and 27 subjects (52%) were nonresponders. The results showing 2% high-level responsiveness to beryllium sensitization in vitro correlate with the 1 to 5% prevalence of chronic beryllium disease in individuals sensitized to beryllium dust in vivo and thus support the thesis that the in vitro immunization system may permit the identification of individuals at risk for beryllium hypersensitivity. Recognized since the second industrial revolution (8, 9, 23), chronic beryllium disease (CBD) is a progressive and poten- tially fatal lung affliction in which inhaled particles of beryllium trigger an immune reaction that inflames the lungs. It is present in 1 to 5% of industrial workers who handle the metal (2, 10, 14, 17, 20, 21). CBD continues to occur in industries such as high-technology ceramics, electronics, dental alloy preparation, nuclear weapons, metal extraction, and aero- space. To immunologists and respiratory biologists, CBD exemplifies unusual cell-mediated immunity and is a model for other granulomatous lung diseases (6). In earlier years, a skin patch test was used to identify some beryllium workers as allergic to beryllium (5). However, the usefulness of the test was limited because evidence of sensitization and the presence of CBD were not correlated and because the test itself could induce beryllium sensitization and thus provoke symptoms in previously healthy workers (18). Beryllium-specific lymphocyte transformation tests (Be-LTT) are not used as in vitro tests for identifying sensitized T cells isolated from peripheral blood or bronchoalveolar lavage fluid of individuals with high levels of beryllium exposure or CBD (12, 20). Current data suggest that the Be-LTT is a specific and sensitive assay for testing beryl- lium sensitization (12, 14, 20). Herein, we describe a new test, the beryllium-specific in vitro immunization system (Be-IVIS), for the generation of beryllium-specific T cells from naive T cells in human peripheral blood. The Be-LTT identifies per- sons who have been exposed to beryllium, whereas the Be- IVIS permits the identification of individuals at risk for beryllium hypersensitivity and provides a useful in vitro human model for studying the immunogenetic, cellular, and molecular aspects of beryllium hypersensitivity. MATERMILS AND METHODS Reagents. Lithium sulfate (Li2SO4), nickel sulfate hexahy- drate (NiSO4- 6H20), barium sulfate (BaSO4), zirconyl chlo- * Corresponding author. Mailing address: Specialty Laboratories, Inc., 2211 Michigan Ave., Santa Monica, CA 90404-3900. Phone: (310) 828-6543, ext. 310. Fax: (310) 828-6634. ride hydrate (ZrOCl2), mercuric chloride (HgCl2), and mag- nesium sulfate (MgSO4) were purchased from Sigma Chemical Company (St. Louis, Mo.). Beryllium sulfate tetrahydrate (BeSO4 * 4H20) was purchased from Aldrich Chemical Co. Inc. (Milwaukee, Wis.). RPMI 1640 medium and Hanks' balanced salt solution (HBSS) were purchased from Irvine Scientific (Santa Ana, Calif.). Ficoll-Paque for in vitro lympho- cyte isolation was purchased from Pharmacia (Piscataway, N.J.). A penicillin-streptomycin mixture was obtained from GIBCO Laboratories (Grand Island, N.Y.), and pooled human AB serum was obtained from Gemini Bioproducts, Inc. (Cala- basas, Calif.). Cell culture flasks (25 cm2) with 0.2-jim-pore- size vented filter caps were obtained from Costar (Cambridge, Mass.). Fluorescence-activated cell sorter lysing buffer, phyco- erythrin-labeled monoclonal antibody (MAb) Leu2a (CD8), and fluorescein isothiocyanate-labeled MAb Leu3a (CD4) were purchased from Becton Dickinson (San Jose, Calif.). An AIS MicroCellector for the selection of T-cell subsets was obtained from Applied Immune Sciences, Inc. (Menlo Park, Calif.). Blood donors. Peripheral blood was obtained by venipunc- ture from healthy human volunteers with no history of beryl- lium exposure. Informed consent was obtained and remuner- ation was provided in accordance with institutional guidelines. A total of 40 to 50 ml of blood was required to initiate the primary culture. Preparation of lymphocytes. Peripheral blood mononuclear cells (PBMC) were isolated from heparinized venous blood by Ficoll-Hypaque gradient centrifugation (4). Cells were washed three times with HBSS, and then their viability was determined by the trypan blue dye exclusion method. Cells were resus- pended in complete medium (20% heat-inactivated pooled human AB serum, 2 mM L-glutamine, 2 mM N-2-hydroxy- ethylpiperazine-N'-2-ethanesulfonic acid [HEPES], 1% peni- cillin-streptomycin [10,000 U/ml]) and were adjusted to 2 x 106/ml. Selection of T-lymphocyte subsets. The AIS MicroCellector cell culture flask system was used for negative selection of either CD4+ or CD8+ cells according to the manufacturer's instructions. In the MicroCellector cell culture flask system, 164 on June 16, 2020 by guest http://cvi.asm.org/ Downloaded from
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CLINICAL AND DIAGNOSTIC LABORATORY IMMUNOLOGY, Mar. 1994, p. 164-171 Vol. 1, No. 21071-412X/94/$04.00+0Copyright (C 1994, American Society for Microbiology
Novel In Vitro Method for Identification of Individuals at Riskfor Beryllium Hypersensitivity
EMMANUEL A. OJO AMAIZE,* MELKON S. AGOPIAN, AND JAMES B. PETER
Specialty Laboratories, Inc., Santa Monica, California 90404-3900
Received 17 August 1993/Returned for modification 27 September 1993/Accepted 14 November 1993
Beryllium-specific lymphocytes were generated by in vitro immunization of peripheral blood mononuclearcells (PBMC) from healthy unexposed individuals. Measurement of blastogenic responses of PBMC by[3H]thymidine uptake demonstrated that sensitization of PBMC with beryllium salts followed by stimulationwith unrelated salts resulted in a negative response, whereas sensitization and restimulation of PBMC withberyllium salts produced a positive response. Flow cytometric and cell depletion analyses showed that all of theresponding cells were CD4+ T cells. The in vitro immunization system was used to screen 52 human subjectsfor susceptibility to beryllium sensitization in vitro. The results show that of the 52 healthy unexposed subjectstested, only 1 (2%) was highly responsive, 4 subjects (8%) were moderately responsive, 20 subjects (39%) werelow-level responders, and 27 subjects (52%) were nonresponders. The results showing 2% high-levelresponsiveness to beryllium sensitization in vitro correlate with the 1 to 5% prevalence of chronic berylliumdisease in individuals sensitized to beryllium dust in vivo and thus support the thesis that the in vitroimmunization system may permit the identification of individuals at risk for beryllium hypersensitivity.
Recognized since the second industrial revolution (8, 9, 23),chronic beryllium disease (CBD) is a progressive and poten-tially fatal lung affliction in which inhaled particles of berylliumtrigger an immune reaction that inflames the lungs. It ispresent in 1 to 5% of industrial workers who handle the metal(2, 10, 14, 17, 20, 21). CBD continues to occur in industriessuch as high-technology ceramics, electronics, dental alloypreparation, nuclear weapons, metal extraction, and aero-space. To immunologists and respiratory biologists, CBDexemplifies unusual cell-mediated immunity and is a model forother granulomatous lung diseases (6). In earlier years, a skinpatch test was used to identify some beryllium workers asallergic to beryllium (5). However, the usefulness of the testwas limited because evidence of sensitization and the presenceof CBD were not correlated and because the test itself couldinduce beryllium sensitization and thus provoke symptoms inpreviously healthy workers (18). Beryllium-specific lymphocytetransformation tests (Be-LTT) are not used as in vitro tests foridentifying sensitized T cells isolated from peripheral blood orbronchoalveolar lavage fluid of individuals with high levels ofberyllium exposure or CBD (12, 20). Current data suggest thatthe Be-LTT is a specific and sensitive assay for testing beryl-lium sensitization (12, 14, 20). Herein, we describe a new test,the beryllium-specific in vitro immunization system (Be-IVIS),for the generation of beryllium-specific T cells from naive Tcells in human peripheral blood. The Be-LTT identifies per-sons who have been exposed to beryllium, whereas the Be-IVIS permits the identification of individuals at risk forberyllium hypersensitivity and provides a useful in vitro humanmodel for studying the immunogenetic, cellular, and molecularaspects of beryllium hypersensitivity.
ride hydrate (ZrOCl2), mercuric chloride (HgCl2), and mag-nesium sulfate (MgSO4) were purchased from Sigma ChemicalCompany (St. Louis, Mo.). Beryllium sulfate tetrahydrate(BeSO4 * 4H20) was purchased from Aldrich Chemical Co.Inc. (Milwaukee, Wis.). RPMI 1640 medium and Hanks'balanced salt solution (HBSS) were purchased from IrvineScientific (Santa Ana, Calif.). Ficoll-Paque for in vitro lympho-cyte isolation was purchased from Pharmacia (Piscataway,N.J.). A penicillin-streptomycin mixture was obtained fromGIBCO Laboratories (Grand Island, N.Y.), and pooled humanAB serum was obtained from Gemini Bioproducts, Inc. (Cala-basas, Calif.). Cell culture flasks (25 cm2) with 0.2-jim-pore-size vented filter caps were obtained from Costar (Cambridge,Mass.). Fluorescence-activated cell sorter lysing buffer, phyco-erythrin-labeled monoclonal antibody (MAb) Leu2a (CD8),and fluorescein isothiocyanate-labeled MAb Leu3a (CD4)were purchased from Becton Dickinson (San Jose, Calif.). AnAIS MicroCellector for the selection of T-cell subsets wasobtained from Applied Immune Sciences, Inc. (Menlo Park,Calif.).
Blood donors. Peripheral blood was obtained by venipunc-ture from healthy human volunteers with no history of beryl-lium exposure. Informed consent was obtained and remuner-ation was provided in accordance with institutional guidelines.A total of 40 to 50 ml of blood was required to initiate theprimary culture.
Preparation of lymphocytes. Peripheral blood mononuclearcells (PBMC) were isolated from heparinized venous blood byFicoll-Hypaque gradient centrifugation (4). Cells were washedthree times with HBSS, and then their viability was determinedby the trypan blue dye exclusion method. Cells were resus-pended in complete medium (20% heat-inactivated pooledhuman AB serum, 2 mM L-glutamine, 2 mM N-2-hydroxy-ethylpiperazine-N'-2-ethanesulfonic acid [HEPES], 1% peni-cillin-streptomycin [10,000 U/ml]) and were adjusted to 2 x106/ml.
Selection of T-lymphocyte subsets. The AIS MicroCellectorcell culture flask system was used for negative selection ofeither CD4+ or CD8+ cells according to the manufacturer'sinstructions. In the MicroCellector cell culture flask system,
IN VITRO DETERMINATION OF BERYLLIUM HYPERSENSITIVITY 165
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1200 -
1000
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800 -
600 -
400 -
200 -
f45 ., " , \
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its \>- <4 Xry:s X S
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10 100 250 500Sensitization Dose of BeSO4 (M)
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FIG. 1. Dose response of naive PBMC from five individuals to sensitization with different concentrations of BeSO4. Normal PBMC obtainedfrom the blood of five individuals were sensitized in vitro to different concentrations (10, 100, 250, 500, 1,000, and 5,000 p.M) of BeSO4 for 6 days.At the end of 6 days, cultures were pulsed for 4 h with I 1iCi of [-3Hlthymidine. The blastogenic response (ACPM) was obtained by the followingformula: cpm for stimulated wells - cpm for unstimulated wells. Each symbol represents one of the five individuals tested. The same symbols areused for the same five individuals here and in Fig. 2, 3, and 4. S.E.. standard error.
MAbs are permanently bound to the surface. When blood orbone marrow samples are added, the immobilized ligands bindto surface antigens of the targeted cells; cells not bearing therecognized antigens remain free in suspension. A quantity of4 x 107 PBMC was added to either the AIS CD4 T-25 flask(MAb Leu3a bound, for depletion of CD4+ cells and selec-tion for CD8+ cells) or the CD8 T-25 flask (MAb Leu2abound, for depletion of CD8+ cells and selection for CD4+cells). After incubation for 1 h at room temperature on a flatnonvibrating surface, nonadherent cells were removed, washedtwice in complete medium, counted, and analyzed by flowcytometry.Flow cytometric analysis of T-lymphocyte subsets. Following
lysis of any remaining erythrocytes, lymphocytes were stainedwith a mixture of MAbs CD8PE and CD4FITC. Stainedlymphocytes were analyzed on a FACScan cytometer (BectonDickinson). Lymphocytes were gated by forward and right-angle light scattering.
Lymphoproliferation. Lymphocytes (2 x 105/0.1 ml of com-plete medium) were dispensed in quadruplicate into 96-wellround-bottom microtiter plates, and antigens in completemedium were added (0.1 ml per well). Nonstimulated controlwells contained 0.1 ml of cells and 0.1 ml of complete medium.Cultures were maintained in a humid incubator at 37°C in anatmosphere of 5% CO2 for designated periods. Four to 16 hbefore harvesting, the cultures in each well were pulsed with 1p.Ci of tritiated thymidine (specific activity, 719.5 mCi/mg; DuPont, Wilmington, Del.). Cells harvested onto filter strips withan automatic cell harvester (SKATRON, Sterling, Va.) were
counted in a liquid scintillation counter (Beckman Instru-ments, Fullerton, Calif.). Data were expressed as changes incounts per minute ± standard errors of the means or as thestimulation index (SI) (cpm for stimulated wells/cpm forunstimulated control wells).
Determination of dose response to sensitization with BeSO4.To determine the optimal dose of BeSO4 for sensitization ofthe primary cultures, a single dose of each of six final concen-trations (10, 100, 250, 500, 1,000, and 5,000 ,uM) was added towells of a microtiter plate containing 2 x 105 lymphocytes in0.1 ml of complete medium. Cultures were incubated for 6days. The blastogenic response was measured as describedabove.
Initiation of primary culture (sensitization). Primary cul-tures of BeSO4-treated cells were initiated essentially as pre-viously reported for other antigens (16). Briefly, 3 x 107 viablecells, in the presence of 100 ,uM BeSO4 in complete culturemedium, were seeded at a concentration of 2 x 106 cells perml in 25-cm2 culture flasks with 0.2-p.m-pore-size vented filtercaps. Flasks were incubated on their flat sides at 37°C in 5%CO, for 10 days. To determine the kinetics of the response tosensitization, cell aliquots (2 x 105 cells each) from 2-day to10-day cultures were placed in wells of microtiter plates andpulsed for 4 h with I ,uCi of [3H]thymidine.
Determination of dose-response curve for secondary re-sponse to stimulation with BeSO4. Following sensitization withan optimal dose (100 p.M) of BeSO4, the sensitized cells wererecovered from flasks, washed three times in HBSS, andresuspended in complete medium. Viable cells, adjusted to 2
FIG. 2. Kinetics of primary response of PBMC from five individuals to in vitro BeSO4 sensitization. PBMC were cultured in flasks for 2 to 10days at a concentration of 2 x 106 cells per ml in medium alone (unstimulated) or in a medium containing 100 F.M BeSO4. On days 2, 4, 6, 8,and 10, 0.6-ml aliquots were taken from each flask, dispensed into six microtiter wells (2 x 105 cells per 0.1 ml per well), and pulsed for 4 h withI p.Ci of [3H]thymidine. The blastogenic response (ACPM) was obtained by the following formula: cpm for stimulated wells - cpm forunstimulated wells. S.E., standard error.
x 106/ml, were dispensed in 0.1-ml volumes (2 x 105 cells per
well) into microtiter wells and challenged with different con-
centrations of BeSO4. The cultures were incubated for 5 days.Four to 16 h before the end of the culture period, the cultureswere pulsed with 1 RCi of [3H]thymidine. Blastogenic re-
sponses were determined as described above.Determination of kinetics of secondary response. Following
sensitization with an optimal dose of BeSO4 (100 ,uM), thesensitized cells were recovered from flasks, washed three timesin HBSS, and resuspended in complete medium. Viable cells,adjusted to 2 x 10"/ml, were dispensed in 0.1-ml volumes (2 x105 cells per well) into microtiter wells and challenged with an
optimal dose of BeSO4 (10 p.M). The cultures were incubatedfor various periods of time (1, 3, 5, 7, and 9 days). Four to 16h before the end of each culture period, the cultures were
pulsed with 1 pCi of [3H]thymidine. Proliferative responseswere determined as described above.
Evaluation of the antigen specificity of in vitro-generatedberyllium-reactive cells. The antigen specificity of in vitro-generated beryllium-reactive cells was evaluated by testing a
battery of related metal salt antigens, including BeSO4. Fol-lowing sensitization of naive PBMC (from five different indi-viduals) to 100 p.M BeSO4 for 7 days in culture, the sensitizedcells were challenged with a single optimal dose (10 ,uM) ofBeSO4, ZrOCl2, HgCl2, NiSO4, Li2SO4, MgSO4, or BaSO4.The level of secondary blastogenic response after 5 days ofculture was measured as described above. As negative controls,nonsensitized cells were cultured in complete medium for 7
days and then stimulated with the different metal salts for 5days.
Establishment of SI cutoff value for determination of level ofresponsiveness. A positive response is defined as a peak SI of-3.0. This value is based on the blood mean peak SI plus 2standard deviations previously established for in vivo sensiti-zation (13). Furthermore, the results of our routine Be-LTT ofmore than 200 beryllium workers and 60 nonexposed subjectssupport this cutoff value.
Screening of 52 healthy subjects with the Be-IVIS. Fifty-twohealthy volunteers between the ages of 20 and 60 were enrolled
TABLE 1. Characterization of in vitro-generated beryllium-specific cells
% of cells expressingT-cell T-cell subset Level of beryllium
" Values are percentages of cells expressing the designated antigens followingdepletion with the AIS MicroCellector cell culture flask system and subjection toflow cytometric analysis.
" Each cell population was sensitized to BeSO4 for 7 days. Following sensiti-zation, each population was either stimulated with BeSO4 or left unstimulated.SI = cpm of stimulated cells/cpm of unstimulated cells.
FIG. 3. Dose-response curves of the secondary responses of beryllium-sensitized cells from five individuals to stimulation with BeSO4. NormalPBMC were sensitized to 100 jiM BeSO4 for 6 to 7 days. Following sensitization, each cell population was challenged with the indicatedconcentrations of BeSO4. S.E., standard error.
in the study (27 males and 25 females). None of the volunteershad ever come in contact with beryllium dust or salts. All 52subjects were screened for beryllium hypersensitivity with theBe-IVIS. A total of five experiments were set up. Ten subjectseach were utilized on separate occasions for four of theexperiments, and 12 subjects were utilized for one of theexperiments.
Establishment of Be-IVIS reproducibility. Three of the 52healthy individuals were tested repeatedly at different timesover an 8-month period for beryllium hypersensitivity with theBe-IVIS.
Statistical analysis. All values are means ± standard errorsof the means. All results are representative data from three or
more consecutive experiments.
RESULTS
Dose response and kinetics of naive PBMC subjected toberyllium sensitization. BeSO4 concentrations of 10, 100, 250,
500, 1,000, and 5,000 ,uM were tested in 6-day primary culturesof naive PBMC from five individuals. The results, shown in Fig.1, demonstrate that 100 ,uM BeSO4 induced optimal levels ofprimary responses in all individuals tested. This concentrationwas routinely used for sensitization. The responses of all fiveindividuals peaked between days 6 and 7 (Fig. 2). None of thefive individuals had an SI of > 1.5 at the optimal time point. Weconsider these responses to be nonsignificant because of theofficial definition of significance (SI of .3) by the Committeeto Accredit Beryllium Sensitivity Testing.
Dose-dependent secondary response of beryllium-sensitizedcells to stimulation with beryllium. The 10 ,uM dose inducedmaximal secondary responses in three of five (60%) cultures ofPBMC from five individuals (Fig. 3). Maximal responses were
induced in each of the other two cultures by BeSO4 concen-trations of 1 and 100 jiM (Fig. 3). These findings support invivo sensitization data demonstrating that a sensitized individ-ual may respond positively to BeSO4 stimulation at either the1, 10, or 100 ,uM concentration (14b).
TABLE 2. Grouping of 52 normal individuals according to level of responsiveness to immunization with beryllium salts in vitro
Group Level of responsiveness Si range No. of individuals % of totalper group/total
a The Committee to Accredit Beryllium Sensitivity Testing has officially defined a beryllium-specific lymphocyte proliferative response with an SI of 3 or greater asabnormal.
FIG. 4. Kinetics of secondary responses of in vitro BeSO4-sensitized PBMC from five individuals to stimulation with BeSO4. Normal PBMCwere sensitized to 100 ,uM BeSO4 for 6 to 7 days. Following sensitization, each cell population was either stimulated with 10 F.M BeSO4 or was
left unstimulated (medium plus sensitized cells only) for 1 to 9 days. The blastogenic response (ACPM) was obtained by the following formula:cpm for stimulated wells - cpm for unstimulated wells. S.E., standard error.
Kinetics of secondary response of beryllium-sensitizedPBMC to stimulation with beryllium. Following sensitizationwith an optimal sensitizing dose of 100 p.M BeSO4 for 6 or 7days and challenge with a single dose of 10 p.M BeSO4, all fiveindividuals tested showed various degrees of positive second-ary responses which peaked on day 5 (Fig. 4).
Specificity of the in vitro-generated beryllium-reactivePBMC. The specificity of in vitro-generated beryllium-reactivecells was demonstrated by the finding that Li2SO4, NiSO4,HgCl2, BaSO4, MgSO4, and ZrOCI2 failed to induce significantsecondary responses in the BeSO4-sensitized cells of all fiveindividuals tested (Fig. 5). On the other hand, as expected fora specific secondary immune response, the BeSO4-sensitizedcells proliferated significantly more in response to stimulationwith beryllium salts (P < 0.01) than in response to stimulationwith the other salts. The fact that comparable responses were
not generated against Li2SO4, NiSO4, or MgSO4 indicates thatthe proliferative response was indeed related to the beryllium
ion and not the sulfate ion in beryllium sulfate. Furthermore,the BeSO4-sensitized cells also responded to stimulation withberyllium oxide (data not shown), supporting the notion thatspecific immune responses can be generated against the beryl-lium ion (14a).
Effect of T-cell subset depletion. After depletion with theAIS MicroCellector cell culture flask system, two-color immu-nofluorescence and flow cytometry demonstrated that all of theresponding in vitro-generated beryllium-specific cells pos-sessed the CD4+ phenotype. No activity was observed in theCD8+ population of T cells (Table 1).
Variation among different individuals in the capacity to besensitized to beryllium salts in vitro. Results of a pilot study ofnine healthy human subjects tested with the Be-IVIS showedthat there might be differences among the subjects in thecapacity to be sensitized to beryllium salts in vitro (Fig. 6). Asa follow-up to the pilot study, 52 healthy human subjects werefurther tested with the Be-IVIS. The results indicated that the
TABLE 3. Reproducibility of Be-IVIS SI results
Subject SI at test date: Average Classificationno. December 1991 May 1992 August 1992
IN VITRO DETERMINATION OF BERYLLIUM HYPERSENSITIVITY 169
1200
1000
800uici+12 6000~
400
200
0BeSO4 BaSO4 MgSO4 NiSO4 L12SO4 HgCI2 ZrOCI2
FIG. 5. Specificity of the in vitro-generated beryllium-reactive PBMC. Normal PBMC obtained from the blood of five responder individualswere sensitized in vitro to 100 ,uM BeSO4 for 7 days and were either stimulated with 10 p.M BaSO4, MgSO4, Li2SO4, NiSO4, HgCl,, ZrOCI2, orBeSO4 or were left unstimulated (medium plus sensitized cells only). The blastogenic response (A CPM) was obtained by the following formula:cpm of stimulated cultures - cpm of unstimulated cultures. S.E., standard error.
52 subjects could be divided into four groups, A through D, onthe basis of the levels of individual responsiveness to secondarystimulation with beryllium (Table 2). Only I of 52 subjectstested (2%) was a high-level responder (group A), whereas 4 of
4
C)
X 3w0z
Z 20
1 2 3 4
52 (8%) were intermediate-level responders (group B). Twentysubjects (39%) were low-level responders (group C), and 27subjects (52%) did not respond (nonresponders) to secondarystimulation with beryllium (group D). Group E included all 52
5 6 7 8 9DONOR NUMBER
FIG. 6. Responses of nine healthy individuals to in vitro sensitization and stimulation with BeSO4. Symbols:stimulated; , BeSO4 sensitized, BeSO4 stimulated.
individuals who served as controls for the secondary stimula-tion experiments, i.e., their lymphocytes were not sensitized toberyllium in the primary culture but were stimulated withberyllium in the secondary culture. As expected, these lympho-cytes did not respond to beryllium (average SI of 1.1 ± 0.3).
Reproducibility of the Be-IVIS. In order to establish boththe interassay variation of the Be-IVIS and the reproducibilityof results used to classify individuals as responders or nonre-sponders, 3 of the 52 subjects were tested three times over an8-month period. The results in Table 3 demonstrate that theBe-IVIS is highly reproducible and that an individual's re-sponse status remains unchanged over time. The interassayvariation was <5%.
DISCUSSION
The increasing use of beryllium in a variety of industriescontinues to be a hazard; in the United States alone, anestimated 800,000 individuals have worked in environmentsinvolving exposure to beryllium or its salts (1). Despite thepotential exposure of many industrial workers to this metal,only 1 to 5% developed CBD (13). Because sensitization isconsidered a predetermining factor for the development ofdisease (7, 13, 21), the need for identification of individuals atrisk for beryllium sensitization cannot be overemphasized.
In the present report, we describe an in vitro berylliumimmunization system (Be-IVIS) for induction of berylliumsensitization in vitro of naive PBMC from nonexposed individ-uals. Our finding that in vitro-generated beryllium-specific cellsare CD4+ T-helper and T-inducer cells is consistent with invivo studies implicating CD4+ T cells in CBD (21).
Because beryllium-specific T cells can be detected prior tothe onset of clinical disease (7, 13, 22, 24), the Be-IVIS allowsdetermination of whether healthy individuals never exposed toberyllium dust or salts vary in the capacity to mount aberyllium-specific T-cell response in a genetically determinedfashion. Our findings on the reactivities of 52 subjects indicatethat variations in the capacity to mount a beryllium-specificT-cell response in vitro exist among different individuals. Thepresent results support our preliminary report on a smallnumber of subjects, which demonstrated that variation insensitization to beryllium in vitro could exist among individuals(15). In support of our findings is the fact that differences insusceptibility to beryllium in two strains of guinea pigs and indifferent strains of mice have been reported (2, 3, 11). Thus,the evaluation of potential genetic markers should be possiblefor individuals identified as responders with the Be-IVIS. HLAlinkage studies indicate that there may be a linkage betweenHLA-DP2.1 alleles and in vivo sensitization to beryllium (19).Extensive prospective studies are required to determinewhether in vitro sensitization might lead to a screening systemto distinguish those individuals who carry the genetic markerand might develop CBD from those with the allele who mightnot become affected.
In conclusion, the Be-IVIS, unlike the skin patch test (5, 18),has potential for gathering information concerning an individ-ual's cellular and immunogenetic responses without disruptingthe individual's immunologic integrity. Successful applicationof workplace screening of all at-risk employees in berylliumplants for susceptibility to beryllium hypersensitivity wouldenable relocation of genetically predisposed individuals so asto minimize or prevent contact with beryllium. Prevention isparticularly important for CBD, a disease for which there is nocure.
ACKNOWLEDGMENTS
This work was supported in part by a grant from the BerylliumIndustry Scientific Advisory Committee.We thank Shirley C. Y. Chang, Michael Archie, and Steve Cai for
their technical contribution; Nancy Campman and Rose Yesowitch forpreparing the manuscript; and Herm Reyes for his critical review.
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