STUDIES ON INCLUSION BODIES
I. ACID-FASTNESS OF NUCLEAR INCLUSION BODIES THAT ARE
INDUCED BY
I N G E S T I O N O F L E A D AND B I S M U T H *
M. WACHSTEIN, M.D.
From the Laboratories of St. Catherine's Hospital, Brooklyn, New York, and the Division of
Pathology, Mt. Sinai Hospital, New York, New York
Inclusion bodies may occur in the cytoplasm of the cell, the nuclei or both.
For some inclusion bodies, as in vaccinia, a virus origin has been established
beyond doubt; for others, particularly those that follow the application of certain
chemical substances, such a causal relationship is unlikely. For the inclusion
bodies which occur after the injection of aluminum and ferric compounds,
Olitsky and Harford23 excluded the possibility of a virus infection by transmission
experiments. Although much work has been done to trace the origin of the
various inclusion bodies to certain normal cellular constituents by various
stains, including histochemical tests, as well as by centrifugation procedures
(Lucas15), the histogenesis and chemical composition of many of the inclusion
bodies is still doubtful.
The present work was first undertaken in order to study a number of histochemical reactions in nuclear inclusion bodies that occurred after the ingestion
of lead in rats. When marked acid-fastness of these inclusion bodies was noticed,
various other inclusion bodies were examined for this property.
MATERIAL AND METHODS
Lead. Eleven young white albino rats of the Wistar strain, each weighing
approximately 100 Gm., were given a diet composed of 59 per cent dextrose, 19
per cent casein, 10 per cent, lard, 5 per cent yeast, 4 per cent salt mixture, 2 per
cent cod liver oil and 1 per cent choline chloride. The diet contained 4 Gm. lead
acetate per kilogram. Two of the animals died after 56 and 92 days, while the
remaining 9 were killed after 142 days. Two control groups of 10 animals each
were given a similar diet which in one instance contained 2.5 Gm. nickel nitrate
per kilogram, and in the other instance contained 1 Gm. cobalt chloride per
kilogram. The animals on cobalt chloride died or were killed after 90 to 95 days.
Bismuth. A block of formalin-fixed human kidney tissue (autopsy # 12933,
Mt. Sinai Hospital) was available for study. This case had been previously used
for the histochemical identification of bismuth in the colon as well as in the renal
inclusion bodies by Wachstein and Zak.31 The patient was a 30 year old white
woman who had a history of rheumatic fever and chorea in childhood, and hypertension two years prior to her death. Her blood Wassermann reaction was 4
plus and she was treated with weekly bismuth injections for one year. She died
two days after a sudden onset of severe dyspnea, and cyanosis. Necropsy
revealed rheumatic heart disease, malignant nephosclerosis and bismuth melanosis.
* Received for publication, January 25, 1949.
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METAL-INDUCED NUCLEAR INCLUSION BODIES
609
Additional material examined. The following tissues containing inclusion bodies were
also available for study:
1. Lungs of a woman with chronic pneumonitis (autopsy #12617, Mt. Sinai Hospital)
2. Salivary glands of a child who died from acute enteritis (autopsy #13389, Mt. Sinai
Hospital)
3. Liver and kidneys of a newborn child (courtesy Dr. P. Gruenwald, Department of
Pathology, Long Island College of Medicine, Brookyln. New York)
4. Beta cells of the chicken pancreas (courtesy Dr. A. M. Lucas, U. S. Regional Poultry
Research Laboratory, East Lansing, Michigan)
5. Intestinal epithelium of mice with epidemic diarrheal disease (courtesy Dr. A. Pappenheimer, Harvard Medical School, Boston)
6. Negri bodies in dog brain (courtesy Dr. J. Schlcistein, Division of Laboratories and
Research, Albany, New York)
7. Bladder of a mink with distemper (courtesy Dr. J. R. Gorham, U. S. Pur Animal
Disease, Research Laboratory, Pullman, Washington)
8. Testicle of a rabbit infected with Virus I I I (courtesy Dr. J. M. Pearce, Cornell University Medical College, New York)
Staining procedures. Sections from formalin-fixed material gave the best
results. Good results were also obtained on material fixed in Zenker's solution,
after the usual treatment with iodine and sodium thiosulfate. The sections were
put in Ziehl-Neelsen's carbol fuchsin at 56 C , for three hours. They were then
washed, decolorized for three to five minutes in acid-alcohol (3 ml. concentrated
hydrochloric acid and 97 ml. of 70 per cent alcohol) and counterstained with
Harris' hematoxylin. Sections were also stained with hematoxylin and eosin
and in addition, special stains were occasionally used.
RESULTS
Lead. In all animals that were given lead, numerous nuclear inclusion bodies
were found in the kidneys; none were seen in the liver. The inclusion bodies
closely resembled those that were first described by Blackman in the kidneys and
livers of children who died of lead poisoning and also in rats that were given lead
in their food.2 Similar observations in experimental animals were later made by
Finner and Calvery, 9 Diaz-Rivera and Horn, 5 and Dalldorf and Williams.4 In
sections stained with hematoxylin and eosin, these inclusion bodies were usually
sharply outlined and homogeneous. Nearly all of them were eosinophilic,
although an occasional one stained purplish. They were round or irregular in
shape and varied in size from tiny droplets to bodies larger than the normal nuclei.
One or several inclusion bodies were found in one nucleus. The large inclusion
bodies were usually centrally located while smaller ones were often found in the
periphery of the nucleus. The nuclei in which inclusion bodies were present
were frequently enlarged. The chromatin was coarse and granular and was
found either located on the outer surface of the inclusion body or had marginated
to the nuclear membrane. Occasionally chromatin was present in both locations
in the same nucleus.
Inclusion bodies were found only in the cortex and in the subcortical zone and
not in the inner portion of the medulla. They were about equally numerous in
the outer zone of the cortex and the subcortical zone. They were found in
tubules that could be easily identified as proximal convolutions but occurred also
610
WACHSTEIN
in tubules forming the medullary rays. The latter predominantly contain
branching collecting tubules and straight thick portions of ascending limbs of
Henle's loops. A more exact localization will have to await the isolation of
single renal tubules as advocated by Oliver.23 (See also Wachstein. 30 )
In addition to the occurrence of inclusion bodies, definite changes were found
in the cortical tubules. Some of the cells showed large swollen vesicular nuclei
as well as degenerative changes in the cytoplasm. These degenerative changes
had progressed in some of the tubules to frank necrosis with sloughing of tubular
epithelium, and frequently to secondary calcification. Some tubules showed a
fiat regenerating epithelium and occasionally mitotic figures. Focal areas of
chronic inflammation were fairly prominent. In all cases, some of the cortical
tubules contained scattered brown-staining granules similar to those previously
described by Fairhill and Miller. 8 In addition, occasional round, concentric
bodies were noticed most often lying within the lumen of the tubules. These
bodies stained brownish and occasionally somewhat bluish in hematoxylin-eosin
preparations. Many of these bodies stained black after treatment of the sections
with cobalt chloride and dilute ammonium sulphide, indicating the presence of
calcium phosphate. They obviously represented partially calcified necrotic
cells. Control sections immersed in dilute ammonium sulphide did not show
any blackening of these bodies or of the intranuclear inclusions or of any other
structures. The absence of histochemically demonstrable lead is in agreement
with Blackman's finding but contrary to those of Finner and Calvery. Basophilic cytoplasmic inclusion bodies described by Diaz-Rivera and Horn were
not seen in our material.
In sections stained by the Ziehl-Neelsen method, the inclusion bodies were
strongly acid-fast and stained red (Fig. 1). There was some variation in the
brilliance of the stain even in sections of the same block. Occasionally inclusion
bodies took a more purplish hue after the counterstain had been applied. This
staining method revealed fine dust-like acid-fast particles in some of the nuclei.
The counterstain clearly outlined the chromatin material surrounding the inclusion bodies within the nuclei. In addition, some of the tubules contained dustlike and occasionally more granular acid-fast material. This apparently corresponded to the previously described brown pigment seen in hematoxylin and
eosin sections, but seemed to be more abundant.
Two unstained paraffin sections from human material that had been collected
by the late Dr. S. Blackman were obtained through the courtesy of Dr. Lucas.
The inclusion bodies proved to be acid-fast although they stained somewhat less
brilliantly than most sections from the fresh animal material. In the control
animals (fed nickel or cobalt) no acid-fast material was encountered. No inclusion bodies or any of the other significant changes were found in the kidneys of
any of the control animals fed either cobalt or nickel.
Bismuth. The cells of the convoluted tubules show inclusion bodies as described by Pappenheimer and Maechling25 within the nuclei and occasionally also
in the cytoplasm. These inclusion bodies were strongly refractile and in hematoxylin and eosin sections had a brownish tinge. They were usually round, occa-
METAL-INDUCED NUCLEAR INCLUSION
BODIES
611
F I G . 1. Section from the kidney of a r a t which was given lead a c e t a t e . Ziehl-Neelsen
stain. Nuclear inclusion bodies stain brilliantly red. Approximately X 900.
F I G . 2. Section from the kidney of a woman who had received weekly bismuth injections
for one year. Xiehl-Neelsen stain. Nuclear inclusion bodies stain brilliantly red.
Approximately X 900.
612
WACHSTEIN
sionally oval, and often occupied a central position in the nucleus. Some of the
nuclei contained several of these inclusion bodies. Degenerative changes in the
tubular epithelium were difficult to evaluate since the kidney, as previously
stated, was obtained from a patient who had had malignant hypertension.
The application of the Ziehl-Neelsen stain revealed that the inclusions were
strongly acid-fast. Both intranuclear and cytoplasmic inclusion bodies stained a
brilliant red (Fig. 2). No other structures were found to retain the carbol
fuchsin after acid-alcohol treatment. The smallest inclusions could be easily
visualized by this staining method.
Other inclusion bodies. None of the other nuclear inclusion bodies that were
examined were acid-fast. These included the nuclear inclusion bodies present in
the salivary glands of a child, as well as those found in liver and kidneys of a
newborn infant; the numerous inclusion bodies present in the alveolar lining cells
of an adult woman who died of pneumonitis; the nuclear inclusion bodies in the
beta cells of the pancreas of the chicken, recently described by Lucas;10 Negri
bodies; the inclusions in distemper, and in the intestinal epithelium of suckling
mice with diarrhea described by Pappenheimer and Cheever;24 or the inclusion
bodies caused by the Virus I I I .
DISCUSSION
Nuclear inclusion bodies have been divided by Cowdry3 into types A and B.
Type A included those which are granular, while type B consist of amorphous,
irregular, masses or hyaline spheres. However, in the study of inclusion bodies,
it is not only necessary to recognize their existence, but also to separate them from
changes that may be caused by the damaging agent itself, on the cellular constituents. By studying their development, from the earliest appearance to the
final stage when the cell dies, one is able to recognize distinct differences in various
inclusion bodies, as has been clearly shown by Lucas. On the basis of studies of
this kind, it seems that in severe burns, yellow fever, and in panleukopenia of
the cat, the inclusion bodies are derived from the oxychromatin which is normally
present in the nucleus but has been increased in amount. 18 In fox encephalitis,
the acidophilic portion of the inclusion bodies seems to come from the oxychromatin surrounding the primary basophilic center.14 On the other hand, in
the case of inclusion bodies in the beta cells of the islands of Langerhans in
chickens, there is no proof for a derivation of inclusion bodies from oxychromatin.
I n the submaxillary gland virus, there is no definite morphologic evidence that
the acidophilic part is derived from chromatin. 28 There is considerable difference
of opinion as to the origin of the inclusions in the herpes, B. virus and pseudorabies group. While Luger and Lauda 19 considered the inclusion bodies of
herpes as accumulated masses of oxychromatin, this is questioned by Lucas and
Herrmann. 17
. In contrast, the inclusion bodies produced by aluminum oxide1 and also those
in lead poisoning13 are supposed to be derived from the plasmosome portion of an
amphinucleolus. I t seems of considerable interest that of ah inclusion bodies
examined only those caused by ingestion of metals proved to be acid-fast. If,
however, these inclusions were derived from the acidophilic portion of the nu-
METAL-INDUCED NUCLEAR INCLUSION BODIES
613
cleolus, a considerable change must have taken place, since the nucleolus itself
is not acid-fast.
The acid-fastness of these inclusion bodies raises the question of the possible
chemical significance of this staining reaction. I t is known that certain cell
constituents may show acid-fastness. A coarsely globular wax-like pigment is
found in the cirrhotic livers as well as other tissues of rats maintained on a low
protein choline-deficient diet. I t was called ceroid.14 Its deposition is strongly
influenced by the amount of cod liver oil given in the diet.29 A similar pigment
occurs also in the smooth muscle cells of the uterus and in other tissues in rats
on a diet deficient in vitamin E. 20 ' 21 In certain nutritional disorders and hepatic
diseases in man, Pappenheimer and Victor26 found similar acid-fast pigment in
various tissues. Acid-fastness has been reported in the lipoid deposits in the
lungs with cod liver oil pneumonia. 10,27 Endicott 7 was able to show that prolonged oxidation of cod liver oil or a linseed oil in vitro with potassium bichromate
renders these oils acid-fast. Linseed and cod liver oil injected into various
tissues of living rats acquire acid-fastness.7, u
The acid-fastness of bacteria and animal tissue is apparently of a variegated
and complex nature. In tubercle bacilli there exists a hydroxy acid, mycolic
acid isolated from the unsaponifiable wax which exhibitis acid-fastness even
after its isolation as a pure substance. On the other hand, acid-fastness in
tubercle bacilli is destroyed after the disintegration of the cellular structure byvarious means which leave the mycolic acid intact. Endospores of bacteria, as
well as human hair retain carbol fuchsin although these substances do not contain
large amounts of lipoids or mycolic acid.
It may be concluded that some lipoid component is responsible for the acidfastness of certain inclusion bodies. Acid-fastness, however, is not the unique
attribute of a substance or a group of substances, but rather is the sign of some
peculiarity of cellular organization related, for instance, to the physicochemical
property of the cell wall (Dubos6).
A certain relationship of some inclusion bodies to lipoid has already been
suggested by the finding of Pappenheimer and Maechling that bismuth-induced
inclusions can be demonstrated by the Weigert-Spielmayer stain for myelin. I n
contrast, however, to the behavior of typical ceroid, the inclusion bodies do not
stain with the usual fat stains, as has already been demonstrated for lead by
Blackman and for bismuth by Pappenheimer and Maechling.
SUMMARY
Various types of inclusion bodies were examined with the Ziehl-Neelsen technic
for acid-fastness. Among those examined, only inclusion bodies produced by
the ingestion of bismuth and lead were found to be acid-fast. Aside from the
possible significance as to their lipoid content, the Ziehl-Neelsen stain is a convenient method for the easy demonstration of these inclusion bodies.
REFERENCES
1. BIRCH, F. M., AND LUCAS, A. M.: Effects of centrifugation on intranuclear inclusions
produced by subcutaneous injections of aluminum oxide. Am. J. Path., 18:1051-1059,
1942.
2. BLACKMAN, S. S., J R . : Intranuclear inclusion bodies in the kidney and liver caused by
lead poisoning. Bull. Johns Hopkins Hosp., 58:384-404, 1936.
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WACHSTEIN
3. COWDRY, E . V . : T h e problem of intranuclear inclusions in virus diseases. Arch. P a t h . ,
18: 527-542, 1934.
4. DALLDORF, G., AND W I L L I A M S , R. R . : I m p a i r m e n t of reproduction in r a t s by ingestion
of lead. Science, 102: 668-670, 1945.
5. D I A Z - R I V E R A , R. S. AND H O R N , R . . C , J R . : P o s t m o r t e m studies on hypertensive r a t s
chronically intoxicated with lead a c e t a t e . Proc. Soc. Exper. Biol, and Med., 59:
161-163,1945.
6. D U B O S , R . J . : T h e bacterial cell in its relation to problems of virulence, i m m u n i t y a n d
chemotherapy. C a m b r i d g e : H a r v a r d University Press, pp. 85-91,1947.
7. ENDICOTT, K . M . : Similarity of the acid-fast pigment ceroid and oxidized u n s a t u r a t e d
fat. Arch. P a t h . , 37:49-53,1944.
8. F A I R H A L L , L. T . , AND M I L L E R , J. W.: T h e deposition and removal of lead in t h e soft
tissues (liver, kidneys and spleen). P u b . H e a l t h R e p . , 56:1641-1650,1941.
9. F I N N E R , L. L., AND C A L V E R Y , H. O . : Pathologic changes in r a t s and in dogs fed d i e t s
containing lead and arsenic compounds; compounds used: lead a r s e n a t e , arsenic
trioxide, calcium a r s e n a t e and lead a c e t a t e . Arch. P a t h . , 27:433-446,1939.
10. G R A E P , I . : Studies in lipid pneumonia; lipid pneumonia due to cod liver oil; lipid pneumonia due to liquid petrolatum. Arch. P a t h . , 28: 613-667, 1939.
11. H A A S , G. M . : M e m b r a n e formation a t lipoid-aqueous interfaces in tissues; a correlation of t h e morphological and chemical aspects. Arch. P a t h . , 28:177-198,1939.
12.
13.
14.
15.
16.
17.
18.
19.
20.
21.
22.
23.
24.
25.
26.
27.
28.
29.
30.
31.
L I L L I E , R. D . , A S H B U R N , L. L., S E B R E L L , W. H . , J R . , D A F T , F . S., AND L O W R Y , J.
V.:
Histogenesis and repair of the hepatic cirrhosis in r a t s produced on low protein diets
and p r e v e n t a b l e with choline. P u b . H e a l t h R e p . , 57: 592-608, 1942.
LUCAS, A . M . : Personal communication t o t h e a u t h o r .
LUCAS, A. M . : T h e cytology of fox encephalitis and t h e effects of centrifugation upon
t h e i n t r a n u c l e a r inclusions. Am. J. P a t h . , 16:739-760,1940.
LUCAS, A. M . : Inclusion bodies. How specific are inclusion bodies for t h e identification of virus diseases? T h e Veterinary S t u d e n t , 4:11-14 and 45-46,1941.
LUCAS, A. M . : I n t r a n u c l e a r inclusions in t h e islands of Langerhans of chickens. Am.
J. P a t h . , 2 3 : 1005-1022, 1947.
LUCAS, A. M., AND H E R R M A N N , W. W.: Effect of centrifugation on herpetic intranuclear
inclusions with a note on cytoplasmic inclusions of unknown origin in t h e r a b b i t cornea. Am. J. P a t h . , 11:963-976,1935.
LUCAS, A. M., AND R I S E R , W. H . : I n t r a n u c l e a r inclusions in panleukopenia of c a t s ;
correlation with the pathogenesis of the disease and comparison with inclusions of
herpes, B-virus, yellow fever, a n d b u r n s . Am. J. P a t h . , 21:435-465,1945.
L U G E R , A., AND LAUDA, E..: Uber oxychromatische Veriinderungen am Zellkern. (Auf
G r u n d von Untersuchungen von Herpes simplex, zoster, Varizellen, Variola unci K a r p fenpocke.) E i n B e i t r a g z u r K e n n t n i s u n d W e r t u n g einschlussartiger Gebilde. Med.
Klin., 22: 415-417; 456-458; 493-497, 1926.
MASSON, K. E . , AND E M M E L , A. F . : V i t a m i n E and muscle pigment in t h e r a t . Anat.
R e c , 92: 33-59,1945.
MASSON, K. E., AND E M M E L , A. F . : P i g m e n t a t i o n of t h e sex glands in Vitamin E deficient r a t s . Yale J. Biol, and Med., 45: 189-202, 1944.
OLITSKY, P . K., AND H A R F O R D , C. G.: I n t r a n u c l e a r inclusion bodies in the tissue reactions produced by injections of certain foreign substances. Am. J. P a t h . , 13: 729748,1937.
O L I V E R , J . : In H a r v e y Lectures. Vol. 40. L a n c a s t e r , P a . : Science Press P r i n t i n g
C o m p a n y , p. 102, 1944-1945.
P A F P E N H E I M E R , A. M., AND C H E E V E R , F . S.: Epidemic diarrheal disease of suckling
mice; cvtoplasmic inclusion bodies in intestinal epithelium in relation t o t h e disease. J". Exper. Med., 88: 317-324, 1948.
P A P P E N I I E I M E R , A. M., AND MAECHLING, E . H . : Inclusions in renal epithelial cells following the use of certain b i s m u t h p r e p a r a t i o n s . Am. J. P a t h . , 10: 577-588, 1934.'
P A P P E N H E I M E R , A. M., AND VICTOR, J . : " C e r o i d " pigment in human tissues. Am. J .
P a t h . , 22: 395-413, 1946.
P I N K B R T O N , H . : T h e reaction to oils and fats in t h e lung. Arch. P a t h . , 5: 380-401,
1928.
ROSENBUSCIL C. T . , AND LUCAS, A. M . : Studies in t h e pathogenicity and cytological
reactions of t h e submaxillary gland virus of t h e guinea pig. Am. J . P a t h . , 15: 303340,1939.
W A C H S T E I N , M . : Influence of cod liver oil on deposition of ceroid in t h e nutritional r a t
cirrhosis. P r o c . Soc. Exper. Biol, and Med., 59: 73-77,1945.
W A C H S T E I N , M . : Nephrotoxic action of d/-serine in t h e r a t ; localization of t h e r e n a l
damage, t h e phosphatase activity a n d t h e influence of age, sex, time and dose. Arch.
P a t h . , 43: 503-514, 1947.
•
W A C H S T E I N , M., AND ZAK, F . G.: B i s m u t h p i g m e n t a t i o n ; its histochemical identification. Am. J. Path.,22:603-611,1946.