Berylliosis As an Auto-Immune Disorder ANDREW L

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Berylliosis as an Auto-Immune Disorder ANDREW L. REEVES, Ph.D. Wayne State University, School of Medicine, Department of Occupational and Environmental Health, Detroit, MI 48201

ABSTRACT Exposure to compounds of beryllium can cause dermatitis, acute pneumonitis and chronic pulmonary granulomatosis (“berylliosis”) in hu mans. These syndromes seem to have an allergic-immunologic component in common. Hypersensitivity to beryllium is of the delayed (cell-mediated) type and can be measured as skin reactivity to patch test; lymphocyte blast transformation; and macrophage migration inhibition. There is good corre lation between the results of these tests in exposed populations, but the degree of hypersensitivity is not necessarily a measure of either extent of exposure or severity of berylliosis. In animal experiment, inhalation expo sure has suppressed a previously established cutaneous hypersensitivity, and degree of hypersensitivity and degree of berylliosis were in significant inverse correlation.

Introduction considerable mortality. It is associated Beryllium is a light metal widely used with inhalation of various, including in in fatigue-resistant alloys, nuclear reac soluble, beryllium compounds, some tors, space vehicle and missile parts, times in very low concentration. Occupa electronics and for other specialized pur tional as well as “neighborhood” cases poses. Exposure to compounds of beryl (the latter having occurred in the general lium has caused dermatitis, acute population living within about a mile pneumonitis and chronic pulmonary from a beryllium plant) are on record; granulomatosis (“berylliosis”) in hu they are collected in a “Beryllium Case mans. The dermatitis is of the allergic Registry.” 13 type with edematous lesions following The allergic nature of beryllium skin contact with soluble salts or with lesions was recognized by Curtis,8 who granulomatous ulceration following the developed a patch test. Concurrently, cutaneous imbedding of insoluble com Sterner and Eisenbud29 noted that the pounds. Acute pneumonitis followed in epidemiology of chronic pulmonary be halation of soluble salts in high concen rylliosis cases also suggested an allergic tration; typical attacks resolved with com etiology. Specifically, it was noted by plete recovery within several months. them that a dose-response relation be The chronic granulomatosis is an insidi tween exposure and incidence was em ous and slowly developing disease with phatically absent, with workers from the 256 BERYLLIOSIS AS AN AUTO-IMMUNE DISORDER 257 cleanest plants and neighborhood cases “tolerogenic”, including a very low dose sometimes showing the worst clinical of beryllium intraperitoneally or a high forms. Furthermore, the morphology of toxic dose intravenously. Krivanek and pulmonary granulomata caused by beryl Reeves18 showed that the beryllium ion lium, with considerable similarity to acted as a hapten in provoking the im those observed in sarcoidosis, also al munological reaction. Complexes where lowed the assumption of an immunologi the beryllium ion was unavailable (aurin- cal etiology. The lesions typically con tricarboxylate, citrate) could not elicit sisted of a cluster of monocytes sur sensitivity, whereas beryllium serum al rounded by a zone of lymphocytes and buminate could elicit stronger sensitivity plasma cells.36 These findings created the than the beryllium ion alone. Vasil’ eva33,34 impression that cutaneous and pulmo detected beryllium-nucleoprotein com nary beryllium lesions may be interre plexes which were antigenic. However, lated, and pulmonary berylliosis may be evidence was also presented that beryl in some way dependent on immune reac lium can interact with cells without prior tions in the organism. complexing to macromolecules and can in Search for humoral antibodies was hibit the response of allergized lympho made24*27,35 and some increases in serum cytes to antigen.15,16 /3- and y- globulin fractions were noted. These results strongly suggested that However, these turned out to be essen the pathogenesis of beryllium disease tially nonspecific. In 1959, it was pointed contained an immunologic component. out that the skin reaction to beryllium The application of this component for being delayed, the immunity is likely to purposes of diagnosis was further be cell-mediated.37 Alekseeva1 and Cirla explored by Epstein and collaborators12-14 et al6 accomplished passive transfer of who first performed lymphocyte blast hypersensitivity in guinea pigs with lym transformation and macrophage migra phoid cells, while the transfer of serum tion inhibition in preparations originat was ineffective. Chiappino et al4'5 were ing from beryllium-sensitive subjects. also able to inhibit all cutaneous reac tions to beryllium in guinea pigs by injec Patch Test tion of an antilymphocyte serum from rab The earliest diagnostic use of im bits. Turk and Polak31 could suppress munologic phenomena in berylliosis was reactivity by intravenous injection of be the Curtis patch test.8 All of the 13 beryl ryllium lactate. Inhalation exposure to lium dermatitis cases reacted positively beryllium sulfate could also suppress to beryllium fluoride. Eight to ten cases cutaneous reactivity.26 Among guinea also reacted positively to beryllium pigs, not all individuals responded iden chloride, nitrate or sulfate. Only three re tically to the beryllium challenge. Ability acted positively to beryllium metal pow to become sensitized was genetically der and none to beryllium oxide powder. controlled and transmitted as a non-sex- However, application of the test ap linked, dominant trait.23 peared to be itself sensitizing and eight Mode of administration and choice of out of 16 controls who have never been beryllium compound also influenced the previously exposed to beryllium com nature of the immunological reaction. pounds experienced spontaneous flareup V acher32 found only those forms and or eczematous reactions at the test site. In routes which were capable of producing a 1955, Sneddon28 reported that a patient complex with skin constituents as im with positive patch test to beryllium de munogenic; freely diffusible forms were veloped a sarcoid-like granuloma at the 258 REEVES test site. Even though Curtis later9 ex Macrophage Migration Inhibition tended his observations to 32 chronic Bloom and Bennett3 in 1966 dem pulmonary berylliosis cases, all of whom onstrated a soluble factor which could reacted positively to the patch test (with inhibit the free migration of macrophages 19 out of 21 control patients reacting in vitro. The factor was elaborated by negatively) it was obvious that extreme sensitized lymphocytes and appeared to caution was advisable in the application be highly antigen-specific but not cell- or of this measure.7’11’20,80,37’38 species-specific. It appeared possible to culture pure populations of lymphocytes Lymphocyte Blast Transformation from buffy coat in the presence of an an Lymphocyte blast transformation can tigen and then test the supernatant of be defined as the morphological en such a culture on guinea pig peritoneal largement of small lymphocytes to large exudate cells. Inhibition of migration of lymphoblasts in vitro.21 The transforma the latter cells was observed if, and only tion can be brought about by numerous if, the source of lymphocytes was a stimuli including tissue antigens, specific sensitized patient This principle was antigens, antisera and a group of very successfully employed with beryllium active stimulants referred to as “nonspe as antigen by Henderson et al,14 who cific” of which phytohemagglutinin demonstrated that beryllium oxide- is the prototype. In 1970, Hanifin stimulated lymphocytes from patients et al12 showed that among specific with berylliosis, but not from normal in antigens, beryllium compounds (includ dividuals, produced the inhibitory factor. ing the insoluble oxide) caused Marx and Burrell19 studied seven patients striking transformation of lymphocytes with chronic beryllium disease by means from beryllium-sensitive subjects only, of the macrophage migration inhibition while the lymphocytes from normal, non test and found positive results in all, sensitive subjects did not transform. while the results in six normal controls Transformation of beryllium-sensitive and two non-berylliotic pulmonary pa and nonsensitive lymphocytes by tients were negative. On the other hand, phytohemagglutinin was similar. In 1973, Jones-Williams17 examined seven other Deodhar et al10 reported on the exploita chronic berylliosis patients and found tion of this discovery for the diagnosis of positive results only in one—but the lat berylliosis. Among 35 patients with ter patients were on corticosteroid chronic berylliosis, 25 (71 percent) therapy at the time of testing. It thus showed at least some blast transformation seems that macrophage migration inhibi (with 21 patients showing strong or very tion is also a useful indicator of beryllium strong transformation), even though these hypersensitivity although it can be patients were treated at the time of the abolished by immunosuppressive treat test with prednisone, an inhibitor of im ment. mune reactions. There was also correla tion between the severity of the clinical Correlation of Test Results disease and the degree of blast transfor Krivanek and Reeves18 as well as mation. Incidence of positive results in Palazzolo and Reeves22 have recently* control groups was low: two out of 28 be examined the correlation of the ryllium workers without the disease; aforementioned methods of assaying de three out of 19 normal healthy subjects; layed hypersensitivity to beryllium in and one out of 11 patients with other lung animal experimentation. In a population of diseases. guinea pigs, frequency distribution of BERYLLIOSIS AS AN AUTO-IMMUNE DISORDER 259

0.5 T

F ig u r e 1. F re quency distribution of the 24-hour response diame ter for control and in jected guinea pigs skin tested with intradermal BeSO-j (0.1 ml of 5 per cent solution).

skin reaction diameters 24 hrs after in- nary or, in severely sick animals, de tradermal injection of 1 y Be (as SO4) crease. In uninjected animals, LW/BW showed about 3.5 mm as the threshold of ratios showed good dependence on both a significant immunologic reaction (fig the length and concentration of beryllium ure 1). This intensity of reaction corres exposure. In intradermally sensitized ponded to about 20 percent migration animals, the LW/BW ratios increased inhibition of peritoneal exudate cells less, and there was negative correlation (figure 2). Skin reaction diameters and between LW/BW ratios and skin reaction percent migration of the macrophages diameters (figure 5) or between LW/BW gave a correlation coefficient of -0.45 ratios and macrophage migration inhibi (figure 3). The correlation of skin re tion (figure 6 ). action diameters and percent blast trans formations has thus far not been quan tified but is also believed to show definite interdependence.10,14 Correlation of Hypersensitivity to Pulmonary Berylliosis Pulmonary berylliosis can be produced experimentally in guinea pigs by inhala tion exposure to beryllium sulfate.25 If the exposed animals were previously sen sitized by intradermal beryllium sulfate injections, it was found that inhalation exposure suppressed cutaneous hyper- sensitively (figure 4). The degree of pulmonary berylliosis acquired by these animals can be quan tified as lung weight-body weight (LW/ BW) ratios because lung weights in pul F igure 2. Correlation of skin reaction diame monary berylliosis, as in most other ters in guinea pigs treated with BeSCh to average pathologic conditions of the lung, tend to percent inhibition of peritoneal exudate cells as increase while body weights stay statio measured by the method of Palazzolo and Reeves.22 260 REEVES

D isc u ssio n Skin testing, lymphocyte blast trans formation and macrophage migration in hibition are methods of assessing delayed hypersensitivity to an allergen. All three methods appear suitable to demonstrate the state of hypersensitivity to beryllium, but since skin testing is an in vivo proce dure believed to cause itself sensitization occasionally, it is best not used on human patients. Among the two in vitro tests of lymphocyte blast transformation and macrophage migration inhibition, it is the latter that has received more intensive quantitative study thus far although both appear suitable measures of delayed SKIN REACTION (mm) hypersensitivity to beryllium. F igure 3. Correlation coefficient (r) and regres It should be emphasized that these sion line for skin reaction diameters of guinea pigs tests measure a state of hypersensitivity treated with BeSOi and percent macrophage migra tion. to beryllium, not a state of berylliosis. The two conditions may or may not go hand-in-hand in exposed humans. In ex perimental guinea pigs, a reverse correla tion was shown to exist. Beryllium in halation suppressed an already estab lished cutaneous hypersensitivity and pathologic response to beryllium inhala tion was milder in sensitized than in non sensitized animals. Inhalation exposure is naturally attended in some measure by trace ingestion of the disseminated com

ES3— 8wk pound. This can have an immunosup Inhalation exposure to pressive effect.2 As for the apparent im 3 0 7 Be (as S0 4 ) / m3 munity to pulmonary berylliosis confer F igure 4. Skin reactivity of guinea pigs to be red by intradermal treatments, the situa ryllium sulfate. tion shows some similarity to the relation of tuberculin sensitivity to tuberculosis, where also a controlled induction of sen sitivity (e.g., with BCG vaccine) is as sociated with increased resistance to tuberculosis. Perhaps the lymphocytic and histiocytic response that follows the induction of cutaneous hypersensitivity stimulates the phagocytosis of inhaled F igure 5. Correlation of skin reaction diame beryllium particles or otherwise helps to ters to lung weight/body weight ratios in guinea pigs with inhalation exposure to BeSOi (14 wk ex destroy the auto-antigen formed in the posure, 35 hrs per wk, to 30 /xg Be per m3). lungs. BERYLLIOSIS AS AN AUTO-IMMUNE DISORDER 261 Conclusions 8- Beryllium is an allergen capable of in ducing delayed cutaneous hypersensitiv 7- ity. Beryllium inhalation causes granulomatous disease of the lungs, the development of which apparently de ? 6 pends on immune mechanisms. The de gree of hypersensitivity can be measured by patch testing, lymphocyte blast trans formation or macrophage migration in hibition. In some but not all cases of human be rylliosis, cutaneous hypersensitivity ac companies the lung disease. It must be 3- o t | t~ | t|— -| - -j ~j i emphasized that the afore-mentioned 10 20 30 40 50 6o 70 So 90100 tests are diagnostic for beryllium hyper % INHIBITION sensitivity, not for berylliosis. In animal F igure 6. Correlation coefficient (r) and regres experiment, degree of hypersensitivity sion line for lung weight per body weight ratios of and degree of berylliosis showed an in guinea pigs treated with BeS04 and percent inhibi verse correlation. tion of macrophage migration. 9. C urtis, G. H.: The diagnosis of beryllium dis References ease, with special reference to the patch test. 1. ALEKSEEVA, O. G.: Inquiry into ability of be Arch. Indust. Health 29:150-153, 1959. ryllium compounds to produce allergy of the 10. D eo d ha r , S. D., Barna, B., and Van delayed type. Gig. Tr. Prof. Zabol. 11:20-25, Ordstrand, H. S.: A study of the im 1965. munologic aspects of chronic berylliosis. Chest 2. Asherson, G. L.: Antigen-mediated depres 63:309-313, 1973. sion of delayed hypersensitivity. Brit. Med. 11. Groetenbriel, C., Van Ganse, W. and Bull. 23:24-29, 1967. O l e f f e , J.: Intoxication with beryllium: 3. Bloom, B. R. and Ben n ett, B.: Mechanism of Pathologic considerations and diagnostic value a reaction in vitro associated with delayed type of the patch test. Acta Tuberc. Pneumol. Belg. hypersensitivity. Science 253:80-82, 1966. 61:363-376, 1970. 4. Chiappino, G., Barbiano di Belgiojoso, G., 12. Ha nifin , J. M., E pstein , W. L., and C line, M. and Cirla, A. M.: Hypersensitivity to beryl J.: In vitro studies of granulomatous hypersen lium compounds: Inhibition of intradermal sitivity to beryllium. J. Invest. Dermatol. reaction in guinea pigs by anti-lymphocyte 55:284-288, 1970. serum. Boll. 1st. Sieroter. Milan. 47:669-677, 13. H ardy, H. L., Rabe, E. W., and Lurch, S.: 1968. United States beryllium case registry (1952- 5. Chiappino, G., Cirla, A. M., and Vigliani, E. 1966). Review of its methods and utility. J. Oc- C.: Delayed-type hypersensitivity reactions to cup. Med. 9:271-276, 1967. beryllium compounds. Arch. Path. 87:131-140, 14. H enderson, W. R., Fukuyama, K., E pstein , 1969. W. L., and Spitler, L. E.: In vitro demonstra 6. Cirla, A. M., Barbiano di Belgiojoso, G., tion of delayed hypersensitivity in patients and Chiappino, G.: Hypersensitivity to beryl with berylliosis. J. Invest. Dermatol. 58:5-8, lium compounds: Passive transfer in guinea 1972. pigs by lymphoid cells. Boll. 1st. Sieroter. Mi 15. Jones, J. M. and Amos, H. E.: Contact sensitiv lan. 47:663-668, 1968. ity in vitro. I. Activation of actively allergized 7. Cotes, J. E.: Discussion of “The role of im lymphocytes by a beryllium complex. Intemat. munologic reactions in pulmonary berylliosis” Arch. Allergy Appl. Immunol. 46:161-171, by Reeves, A. L., Swanborg, R. H., Busby, E. 1974., K., and Krivanek, N. D. Inhaled Particles III, 16. Jones, J. M. and Amos, H. E.: Contact sensitiv (Walton, W. H., ed.) Old Working, Surrey, Eng ity in vitro. II. Effect of beryllium preparations land, Unwin Brothers, p. 608, 1971. on the proliferative response of specifically al 8. Cukns, G. H.: Cutaneous hypersensitivity due lergized lymphocytes and normal lymphocytes to beryllium. Arch. Dermatol. Syphilol. stimulated with hemagglutinin. Intemat. Arch. 64:470—482, 1951. Allergy Appl. Immunol. 48:27-29, 1975. 262 BEEVES

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