To clarify immunopathological mechanisms in granulomatous hypersensitivity (GHR) to beryllium (Be), migration inhibitory factor (MIF) was assayed. Blood lymphocytes from three patients with GHR to Be and two normal persons were isolated and cultured with and without BeO or other antigens. Cell-free supernatants removed daily were dialyzed, lyophilized and assayed for MIF by measuring the area of migration of normal guinea pig peritoneal exudate cells out of capillary tubes within 24 hours after exposure to the supernatant. BeO added to sensitized lymphocytes produced supernatant that decreased migration, in contrast to supernatant from non-sensitized lymphocytes, indicating that BeO-sensitized lymphocytes cultured with Be elaborate a soluble factor, MIF, which correlates with delayed hypersensitivity and may play a role in granuloma formation. It may also prove useful in diagnosis of berylliosis

Epstein, William L.

Fukuyama, Kimie

Henderson, William R.

Spitler, Lynn E.

Elsevier - Publisher Connector 

THE .Jo1 tuo:.u. m lNV&."'TU:A11\ ~ OutMATOl..()(,'1 
('npvn~ht ~ 1972 h' l"ht \\ olhom• I. \\ tlkm• C'u 
Vol 58. '\o I 
Prtnted an U.S.A 
IN VITRO DEMON TRATION OF DELAYED HYPER ENSITIVITY IN PATIENT 
WITH BERYLLIOSIS* 
\\'lLLIAM R. HE)JDER. 0:\tt. KIMlE FUK UYAMA. M.D.t. WILLIAM L. EP TEl)~, M.D.t 
A~O LY~:-.1 E. SPITLER. M.D.t§ 
AB TRACT 
T o clarify immunopathological mechanis ms in granulomatous hypersensitivity (GHR) to 
beryllium (Be), migration inhibitory factor (MIF) was assayed. Blood lymphocytes from 
three patients with GHR to Be and two normal persons were isolated and cultured with 
and without BeO or other antigens. Cell-free supernatants removed dai ly were dialyzed, 
lyophilized and assayed for MIF by meas uring the area of migration of normal guinea pig 
peritoneal exudate cells out of capi llary tubes within 24 hours after exposure to the super-
natant. BeO added to sensitized lymphocytes produced supernatant that decreased migra-
tion, in contrast to s upernatant from non -sen;,itized lymphocytes, indicating that BeO -
sensiti zed lym phocvtes cultured with Be e laborate a soluble factor, MIF. which correlates 
with delayed hypersensitivity and may play a role in granuloma formation. It may also 
prove useful in diagnosis of berylliosis. 
Delayed type hy per ensiti,·ity can be demon-
s trated in patient~ with berylliosis by mean;, of 
skin tests in uiuo ( 1) and lymphocyte transforma-
tion in uitro (2). The rela tionship of delayed hy-
persensitivity to induction of granuloma. how-
ever. remain;, unclea r. To funhe r elucidate immu-
nopathological mechanis ms in berylliosis. we as-
sayed the induction of one mediator of cellular 
immunity, macrophage migration inhibito ry 
factor ( MIF), by the method of Rocklin et al. (3). 
By this method. peripheral blood lymphocy tes 
from sens iti zed pa tients, when incubated with 
s pecific antigen in uitro. produce MIF which in-
hibits the migration of normal guinea pig perito-
neal exudate cells. 
MATERIALS AND METHODS 
'ub;ect., Three patients with localized. expenmen-
tally tnduced. granulomatous ;.km lesions of berylliosi~ 
and two normal ~ubjects "ere s tudied in !his expen-
mem Oelayt>d hyper..en~itivit\ reactions (DHR) to be-
ryll ium ;,sits wen:> determined by patch testing with 
0.1 o, bervlltum fluoride t He r ) nr 1.0": bervllium sulfate 
(Be SO ,l. and granulomatous response was detected by 
tntradermal InJections of 10 p.g 1.0 p.g beryllium oxide 
<Be0 ) (4) Subjects were abo sktn tested for DHR to 
PPD (purilied protein derivative of tuberculin) and 
SKSD btreptokmase-streptodornase) . 
Lymphocyt e i.~olatwn and MlF inductiOn. Lympho-
cyt e~ from these donors were ISOlated by incubating 
plasma obtained from 50 to 100 ml blood on glass wool 
<·olumns to remove adherent phagocytic cells (51. After 
Hanks' BSS washing. cell preparations consisting of 
94<11 lymphocytes were distributed into quadruplicate 
- .- From the Uni,ersity of California School of Medi-
cine, Departments of Dermatulogyt and Medicine,t an 
F'ranc1sco Med1cal Center. San Francisco. California 
941 22 
§ Rec1p1en1 of a Dernham Fellowship CD-161! from 
the Californta Division of the American Cancer Societv. 
Rece1ved ,July I:.!, 1971: accepted for publication Sep-
tember 9, 1971. 
This study was supported in part by ~alional Insti -
tutes of Heahh grantti .¥A MO 5372. AMO 7939 and A1 -
09461. 
5 
sets of culture tubes. Ui ml/ tube. at a concentrat ion of 
a 10' cells/ ml TC 199 medium without serum or anti-
biotics t:ll. The tube" were incubat~>d for :l days at 37 (' 
tn an atmn~phere ol 9fi"r air and 5'1 CO, wtth and 
without the test amigen BeO t 1.5 p.g/ mll. To insure reli-
abtlitv ol the test , PPD ( 10 p.g/ mll and KSD (50 
units/ mil were also used as antigens for induction of 
MIF. The cell free supernatants were removed each day 
during the period and the medium. with and without 
the original antigen. was replaced to the original volume 
in the appropriate tubes. Supernatanrs obtained from 
each tube were pooled. dialyzed in the cold against 0.15 
ml NaCI and theo al(ainst dis tilled water for a total of 
48 hours. and concentrated by lyophilization. For test-
ing. the lyophilized ~upernatant was made up to a 5-fuld 
concentral ion of the original volume of 16 ml using TC 
199 with antibiot ics. 10'', normal heat inactivated 
I(Uinea pig serum. and 5": fetal bovine serum. Induction 
of M IF' was performed twice in separate tr ia l~ for each 
subject tested. 
Macrophaf!e m&~ratwn mhibitio11 as~a\. Peritoneal 
exudate cell" (PECI from normal I(Uinea pigs were in -
duced by tntraperitoneal mieclion of light mineral oil 
and collected in unsiliconized glassware. The cells were 
washed in Han k ' B ' S and suspended in TC 199 con-
tainmg anltbtoltcs. These suspensions containing mac-
rophages and lymphocytes were drawn into capillary 
tubes and packed by centrifugation. The capillaries 
were cut nt the cell -lluid interface and the cell -con-
taming portion was placed on !he bottom coverslip of 
Mackaness type chambers. The chambers were then 
covered wilh a second coverslip and filled with the me-
dium to be assayed. Each sample was prepared with a 
minimum of four capillaries. After 24 hours' in<'uballon 
!II :n• C. the area of macrophage migration was pro-
jected and mt>asured by planimelry. l n calculating the 
experimental data. the following formula was used: 
~, migrat ion 
area of migratton in supernatant with antigen 
art>a of m il(ratton in ~upernatan! without antigen 
" 100 
The assay was usua llv performed two to three times 
using macrophages from different guinea pigs. 
6 THE JOURNAL OF INVESTIGATIVE DERMATOLOGY 
RF.Sl!LTS 
The Figure shows photomicrngraphs of normal 
guinea pig macrophages which migrated out of 
capillanes m the presence of supernatants ob-
tamed by mcubatmg lymphocytes from one 
normal person (Normal #l) and from one patient 
(Patient A) with test antigens BeO. PPD and 
SKSD. Decreased migration areas are seen when-
ever macrophages were mcubated with a speciftc 
antigen to which the subJect had positive delayed 
skin test reactions. Thus lymphoc~·tes of Patient 
A. who had positi\e skin tests to all three anti-
gens. produced MIF when stimulated bv each ot 
the three antigens. In contrast, the ly~phocvtes 
of subject :--1, (:--lurmal # l). who wa!'l negative to 
the ReO skin test, dtd not produce MIF when 
stimulated by HeO. This finding ruled out the 
possibility that the concentratiOn of BeO used in 
this experiment tould be nun-~pecifically c~to­
wxic to guinea ptg macrnphages. On the other 
hand. hts lympho<·vtes did prndu<·e YIIF onlv 
when sttmulated hv anllgens IPPD and SKSDI 
which gave positl\'1.' skin I est reactions m L'WO. 
The quantitalt\1.' results for '( macrophage mi-
gration obtained bv plammeter measurement are 
tabulated (see Table). Lymphoc.vtes trom BeO 
skin test poslttve indivtduals (Patients S. A. Bl 
produced MIF which resulted in migratinn areas 
of 48cr. 75c-; and 59'7r of cuntrols. 
In contrast. migration areas of 105'·, and 9:l' r of 
controls were obtained when macrnphages were 
incubated with supernatants from BeO stimu-
lated lymphocytes from normal individuals IN 1 
and al. Thus the mean rnigratl()n was 58.5'1 ± 
17.0Dif for the three BeO positive individuab. 
whereas the mean migration for the two BeO neg--
ative individuals was 97.0", ~ 6.9(',. This differ 
ence (by t test) was significant at p <.. 0.001. All 
except Patient B were !'kin test positive to the 
other antigens studied m thts experiment. PPD 
and SKSD. Migratwn area was decreased to av-
FtGl'RE. Macrupha~e mtgrution inhibition. Compare 
area ol mtgralton with control. In the normal inhibition i.~ seen \\ith PPD and SKSD: tn the patient ~•ilh henl-
liol>is. it is seen wtth BeO PPD and SK::iD 
TABLE 
---
lit· !I I' I'D ~KSil 
~uh 
,... .. 
..... "I " ... ~kUl Mu!r '''"~ "'"  ~, 'ftll ,,.,, ·~· lf>1ol Urf11 
s .. !l6. f>:l. :ll + lib .. '):!. 19 + :) 1. 71. .')I 
\ 7:!. i9 + fll. -~9 + 6~. 71 
B Hi. 7:1 t 6·1. I I:! 
'· 
w.; fi:l fill 
'· 
j ~.1. 9:1 I - it 71 J - I AA. 6:! 
erage of 7:3' <, 55•·;, 85'·c. 5:J<t and 7:n with PPD 
and of ;>R''(, 7l't. 60', and 6.')Cf with SKSD. 
OISCTSStON 
The results d~>mnnstrate that ReO-stimulated 
lymphocyte~ from patients with berylliosis, a 
metal induced granulomatous disease. produce in 
vitro :v1 IF. a factor whit•h inhibits migration of 
nnrmal ~rutnea ptg macrophages. The production 
ul MIF' 1:-. immunologtcally specific. smce BeO 
does not ~ttmulate lymphocytes from normal tn· 
dindual!' to produce MIF. The induction of MIF 
111 sensitized indi\"iduals constitutes an m uttro 
assav for delayed hypersensitivity in patients 
with henllio;;is . 
Development of m uttro methods has provided 
tools to mve~tigate the mechanism~ of delaved 
h\'persensttJvity. The original observattons of 
eeorge and Vaug-han 161 on migration inhibition 
were extended by David (7). who showed that the 
migration of tells rrom guinea pig-s with de laved 
hypersensitivity to tuberculin was consistently 
and markedh· inhibited to ph~·siological concen' 
trations of PPD. Unrelated antigen had no effect 
on macrophage migration. When these sensiti'e 
n•lls were diluted with normal cell:-. to a~ low as 
2N' of the total. then the entire population in-
cluding the 97.fi~, normal cells was inhibited h~ 
anttgen (8) · 
Bloom and Bennett demonstrated that mhibt-
tton ol migrat1on 1s mediated bv a soluble factor 
MIF. which is released by lymphocytes from sen: 
sitized donors upon contact with specific antigen 
(9). MlF is non-dialyzable and heat stable at 56 
C' for :Hl minutes. MIF acti,·ity is destroved bv 
chymoi T) psm and neuramindase hut is i-nsensi-
tive to RNAase and D. Aase (10). MTF elutes 
from Sephadex G I 00 in a fraction after the al-
bumin peak, its peak activity in sucrose gradient 
centrifugation bein~t approximately at :15. It mi-
grates in disc electrophoresis at pH I .1 anodally 
to albumin and has a higher buoyant density than 
pure protein (11). These experiments suggest that 
MIF is an acidic J!l:vcoprotein with a molecular 
weight of about 40.000. 
Production of MIF is inhibited by puromycin 
( Ill . suggest mg that stimulated lymphocytes ac-
tively synthesize matenal(s) in response to an-
tigen and do not merely release a pre-formed 
factor. InjectiOn of purified preparations con· 
MIF AND BERYLLIOSIS 7 
tammg MIF into the skin of normal guinea pigs 
provokes induration and erythema similar to a 
delayed sktn reaction ( 1~). 
Studies by Rocklin and Thor (3. 13) showed 
that MIF production occurs on ly when the subject 
exhibits delayed hypersensitivity to the stimu-
lating antigens. such as PPD. histoplasmin. cocci-
dioidin. candida a lbicans and SKSD. SKSD in-
duces MIF in almost all normal subjects tested 
and is considered a suttable a ntigen to test for 
aneq~y. The human MIF' assay method has been 
used successfully for detect ion of cellular semd-
tivity or its lack in glomerulonephritis, sarcoi-
dosis and Hodgkin's disea!le (14). The results were 
reproducible and correlated with in uivo tests for 
cellular hypersens itivity .. The in vitro MIF assa:-, 
is considered reliable for cltnical investigation of 
delayed hypersensiti\ itv (:l). 
Evidence suggests that berylliosis is a disease 
associated with delayed hypersensitivity (DHRl 
to beryllium (I, 15 17). similar to the findings in 
tuberculosis: and. as in tuberculosis. the role ot 
DH R tn the pathogenesi~ of granuloma for malton 
in berylliosis rematns obscu re ( 18). Until more 
\\ork i;. done with granulomatous diseases lacking 
delayed sensitivity. such as sarcoidosis and zir-
conium granulomas !4. 18), the present findings ot 
MIF in berylliosis must he considered a measure 
of D HR only and not ol granulomatous hypersen-
sitivi ty. 
Be exposure orcurt- primarily in workers en-
gaged in beryllium processing. but rarely affect::; 
skin except by di rect trauma ( 18). In about 50(', of 
cases collected by the Beryllium Case Registry 
( BCR). the patient developed symptoms while on 
the job (19). but the latent period varied widelv. 
up to 20 year~ from exposure to onset of clinical 
disease. Bv 1966. ;,:~5 cases of chronic berylliosis 
had been recorded in the BCR: the true incident•e 
is likely mu<·h higher, since the disease frequent!~ 
goes unrerogntzed. 
Clinica l dtagno::.ts ot pulmonary heryllio:<is can 
be difficult. Histologtcally lung section:. shO\\ a 
diffuse interstitial non-caseating granulomatous 
pneumonitis (20) , often indiMinguishable from 
sarcoidosis. Patch test!- \\ ith Be salts and the 
Kveim test have been used to differentiate bervl-
liosis from sarcoidosis. Generally, the K vei m test 
is negative in berylliosis, whereas Be, 0, patch 
tests are negative in sarcoidosis. The high inci -
dence of false positive K veim tests reported by 
Israel a nd Goldstein (21) in patients with per-
s is tent adenopath) of diverse causes requires con-
firmation before the K veim test is abandoned. A 
lung biopsy for histological and metal analysis is 
needed to unequivocally establish a dial{11osis. 
The use of patch testtng has several drawbacks. 
Despite negative results. several patients have 
been cons1dered to suffer from berylliosis on the 
basis of history, clinical findings and lung biopsv 
analysis (15). ' kin tests may affect the immuno-
logical s tatus. possibly leadmg to contact sensni-
zation and an altered response to inhaled Re. 
Exacerbations of respiratory symptoms have been 
reported following patch tests (16, 17). Since BeF 
is a potent sens1l1zer, patch testing would be 
contra -indicated for possible pre-screening for 
beryllium sensitivity. 
In t'L lro demonstration of delayed hypersensi -
tiVIty to bervlltum has been reported recently. 
Lymphocyte transformation by BeO has been 
observed in patient!:i with berylliosis !2). There is 
some questiOn, however, about the specificity of 
lymphocyte transformation. since PHA nonspecif-
ically induces blastogenic transformation and the 
correlation with delayed hypersensitivity is im-
perfect ( 22). Our 1n vitro demonstration of M IF 
production in patients with cutaneous berylliosis 
may prove a useful model in the s tudy of pulmo-
nary berylliosts. This assay could he used as a 
screening test for workers exposed to high leveb 
of Be tn the van ous industries where beryllium i!> 
processed and used for manufacturing purposes. 
A change of tmmunolog1cal status from a negative 
to a positive assay for MIF would signal a need to 
limit the worker\ future exposure to beryllium. 
possibh to decrease or prevent progression to 
dinical disease. The test also should prove useful 
Ill screening patients suspected of ha\ ing sarcoi-
du::.ts. 
RF.FERE~CES 
1. Curtis. (; II Cutaneou;, hyperhensitt\ tty dul' to 
hNyllium Arch. Dcrm Syph .. 6·1: ·riO. 19.1 I. 
·> ltonifin. ,J. i\1 .. Ep~tc1n. \\" L. and Cline. M .. J. ln 
t•ttro ~tudtcs of granulomatous hypersensitivit~ to 
hE>ryllium. ,) Invest. Derm .. 55· 281. 1970. 
:1. Hncklin. R E .. Meyers, 0. L. and David .. ). R.: An 
rn t•rlro a~sa\ for cellular hypersensiti\·i ty in man 
.J lmmunul.. /()./ I. 1970. 
t. Ep~tem. \\' L .. Metal-induced ~rranulomatous hy-
pc,.,ensillvll\ m man. p. :ll:t /mmunologv and 
th1• Skin. Eds .. Mnnta~na, \\'. and Billingham, R 
E. Am>leton-Centur'> Crofts. :o;e" York. 1971 
!i. Cline, M .J and Swett. \ C : The mtcractlnn ot 
human monontes and lymphocytes .. ) F.xp 
Mcd. /28. t:I09, 1968. 
fi. C!'nri(P. M and \au~han .. J H .. In trtro cell mtgra-
ltcm <l» a mndel Jnr de laved hypcr .. ensill\ ll'. Proc. 
Soc Exp Bioi. Med .. Ill 51-1, 1962. 
7 Dovtd. ~J R ~ Al-Askan. S .. La\vrence. H ~. and 
Thomas. 1.. 0l'laved hype~ensillvtt) in t•ttro. I. 
!'he sp<'t'lfit'tlY of inhibition of cell mi~rration h) 
anttgcn,. ,J lmmunol.. 9.'1- 26-1. 196·1. 
~. Davtd .. ). It, l.a\\rence, H. S. and Thoma~. L.: Oc-
l,lyed hypersPnsllivtt\ in utro. II. Effect of scn,.i-
ll\e cells on normal cells in the presence of an-
tt!(cn .). lm munnl.. 9.1 274, 196·1. 
!!. Blt~)m. B R. and Bennett , B.: Mechani"m of a re-
at·tion in t•1tro associated with delayed type hy-
persenstti\ ity. Science, 153.· 80. 1966. 
10. Remold. H. G .. Kat1., A. B .. Haber, E. and David .. ). 
H. : Studies on migrat1on inhibitory factor (M IFI: 
Hecuven or MJF activitv after purification hy ~el 
ftltratwn and di,c electrophore is. C'ell. lm -
munnl.. I l:l:l. 19i0. 
II. Oand .. ) R. Dela~pd hypersensitivity m v11ro. Its 
m!'d1at1on h\ l'ell frpe substances formed bv 
lymphoid t:ell anugen interaction. Pruc 1'\at 
Acad Sci ... 56. I. 1966. 
12. Lawrenre. H S. and Land\, M. : Mediator~ of Cel-
lular lmmunll\ . Academic Press, · ew York. 1969. 
1:1. Thor 0 .. ,Jurezt~. R E .. \each. S. R., Miller, E. and 
Drav. S .. Cell mt!(rauon Inhibition factor released 
8 THE JOURNAL OF INVESTIGATIVE DERMATOLOGY 
bv anttgen from human penpheral lymphocytes. 
:-.lature. 219.· 755. 1968. 
11. Rocklin. R. E., Lewis. E . • 1 and Da\·id. J R In 
ttlro ev1dence for l'ellular hvpersensiti\'it~· to 
glomerular basement membrane an tigens tn 
human glomerulonephritis. :'~Jew En!( .. J. Med .. 
28.'1' 10, 1970. 
15. Curt is. G. H.: The dial(nosis of beryllium disease. 
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16. Sneddon. I. B.: Ber\lliol'i!i. a ca~e report. Brit Med. 
.J., I 14-18. 1955. 
17. Waksman, B. H.: The dta~mosts of ben· Ilium diseal-.e 
with special reference to the patch test. Arch. 
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18. Epstein, \\'. L.. Granulomatou!' hypersensitivity 
Progr. Aller!( .. II. :16, 1967. 
19. Stoekle. J. 0 .. Hardy, H. L. and \Veber. A. L.: 
Chronic beryll1um d1sea~e. Long-term follow up of 
six tv case and selective review of 1 he literature. 
Amer. J. Med .. 46.· 545, 1969. 
20. Chloak, .1.: The anal.vsi!' of traces of beryllium . Arch. 
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I 
1 


In Vitro Demonstration of Delayed Hypersensitivity in Patients with Berylliosis 

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In Vitro Demonstration of Delayed Hypersensitivity in Patients with Berylliosis

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