Chronic Respiratory Disease in BALBIc Mice

15 Pages·2007·4.7 MB·English

Chronic Respiratory Disease in BALBIc Mice
I. Pathology and Relation to Other Murine Lung Infection
P. Ebbesen, M.D.
CHRO-NIC RESPIRATORY DISEASE iS a common finding in long-
term experiments with mice of several strains. The work of Nelson
has shown that the disease embraces at least two different entities:
infectious catarrh caused by Mycoplasmra pulmonis1'2 and chronic
murine pneumonia or enzootic bronchiectasis probably caused by a
virus.2,3
In experiments with a murine subeellular leukemic agent inducing
plasma cell leukemia, a chronic respiratory disease was noticed in old
females." Since BALB/c mice are very susceptible to chronic lung
infections,7 an investigation of the disease in this strain was undertaken.
Material and Methods
Inbred BALB/c mice obtained in 1963 from the National Institutes of Health,
U. S. Public Health Service, were used. Infectious material was obtained from
old untreated BALB/c females with spontaneous pneumonia.5 Two consolidated
median lung lobes and a consolidated lower right lobe in 2 ml. saline were crushed
in a Potter-Elvehiem type homogenizer, spun at 1000 g for 20 min., and the
sedimentdiscarded.
For transfer experiments, 20 pJ. of this standard suspension were inoculated in
each nostril of ether-anesthesized mice; controls with each experiment included
mice inoculated with normal lung tissue and untreated mice. Filtration of the lung
suspension was performed with Millipore filters (Bedford, Mass). Before filtration
through 450 and220m,u, Bact. prodigiosus was added, and before filtration through
100 m,u herpes virus was added to the suspension, and samples of filtrate were
cultivatedforthe appropriate microorganisms.
Ether inactivation was attempted by miing 1 part of ether to 4 parts of standard
lung suspension for storage at 40 C. overnight. Heating of lung suspension to 560 C.
for 30 min. was carried out in a water bath. Agar double-diffusion was performed
by the method of Wadsworth.8 A supernatant of the usual lung suspension, ob-
tained after centrifugation at 10,000 g for 20 min., was used as antigen, and
serum from mice with lung disease as the source of antibody. Single radial diffusion
From the Department of Biochemistry A, University of Copenhagen, Copenhagen,
Denmark.
Supported in part by grants from the Anders Hasselbalchs Fond til Leukemiens
Bekaempelse, the Carl og Ellen Hertz's Legat til Dansk I-ege og Naturvidenskab, and the
DanishState ResearchFoundation.
Accepted forpublicationApr. 1, 1968.
Address for reprint requests: Dr. Ebbesen, Department of Biochemistry A, University
of Copenhagen, Copenhagen, Denmark.
* The cultivations were kindly carried out by Dr. Riewerts Eriksen and Dr. P. R.
Hornslet, Institute ofMedical Microbiology, UniversityofCopenhagen.
219
220 EBBESEN Vol. 53, No. 2
was performed, according to the method of Mancini et al.,9 with agar and standard
lung suspension mixed 9:1 and 1:1 and with serum from sick mice in the well.
Hemagglutination was carried out by incubating 0.5 ml. of a supernatant
obtained by centrifugation of the standard lung suspension at 10,000 g for 20 min.
with an equal volume of red blood cells which had been washed three times and
made into a 0.5% suspension in saline. The test was read macroscopically after
2 hr. at 370 C. Inhibition of agglutination was attempted by incubating lung
suspension with antiserum for 30 min. at 370 C. before adding the cells. The
antiserum was from mice with lung disease and from rabbits; the antiserum was
immunized with lung suspension and absorbed with washed red blood cells from
untreated mice. Agglutination was further attempted with a modified Coombs
test10 usingredbloodcellsfrommicewithpneumonia andtherabbit antiserum.
Cold agglutinins were sought by the technique of Smadel.11 As a test for
neutralizing antibodies, equal volumes of hmg extract and serum were mixed and
incubated at 370 C. for 30 min. and subsequuitly inoculated intanasally. The
serum was from BALB/c mice with and without the lung disease and from Swiss
micewith andwithout greylungdisease.12*
Mice showing signs of illness were killed shortly before expected death; the
remaining mice, 8 months after inoculation, and necropsy was done. Blood was
taken for electrophoresis on cellulose acetate strips.1" Leukocyte counts were made
and hematocrit values determined. Lung, liver, spleen, kidney, peripheral lymph
nodes, thymus, and thyroid were taken for microscopic study. Sections were stained
with hematoxylin and eosin and the periodic acid-Schiff (PAS) reaction. In selected
cases, diastase-PAS, thioflavine T, and alkline Congo red14 were used in connec-
tion with polarization optical examinations." Staining for elementary bodies was
done with a modified Gram and a modified Giemsa stain.'6 The degree of amy-
loidosiswas estimatedonsections ofthespleen and gradedfrom 1 to6.1'
Results
Intranasal inoculation of extract from diseased lungs resulted in death
of the recipients in pneumonia and spleen amyloidosis after 2-6 months.
Repeated passages with lung tissue from normal mice caused no lung
lesions and pneumonia was not observed in the untreated controls.
Amyloidosis of the spleen did not occur in controls killed at the age of 5
months, but small amounts were found after 8 months in some controls
(Table 1).
Killing of preselected inoculated mice after 1 week revealed infiltra-
tions of small lymphocytes around bronchi (Fig. 1 and 2) in the lower
right lung lobe (Table 2). On the fourteenth day both right median
lung lobes were firm and yellow, with lymphocytes around bronchi and
bronchioli, granulocytes intrabronchially, and collapse of many alveoli.
The main bronchi to the median lung lobes showed no sign of compres-
sion. At the thirtieth day, chattering was often heard from the otherwise
healthy-looking mice. Now both the median and the lower right lobes
were consolidated. The bronchioli were filled with granulocytes (Fig. 3)
' The latter serums were a gift from Dr. R. H. Leach, the WeIlcome Research
Laboratories, London.
August 1968 RESPIRATORY DISEASE 221
Table 1. Induction of Pneumonia and Spleen Amyloidosis in BALB/c Mice by Intra-
nasal Inoculation ofLung Extracts
Mean Meangrade*
survival ofspleen
lnoculum Sex Pneumonic/Total (days) amyloid
Pneumonic lung M 20/20 110 1.7
F 20/20 86 2.0
Normal lung M 0/10 150 0
F 0/10 150 0
M 0/10 270 1.0
F 0/10 270 0
None M 0/10 150 0
F 0/10 150 0
M 0/10 270 0
F 0/10 270 0
The degree of amyloidosis was graded from 1 to 6 according to the meod of Christensen
etWIY
Table 2. Development of Clinical and Pathologic Lesions in Experimentally Induced
Chronic Respiratory Disease in BALB/c Mice
Day" Clinicalstatus Organ lesions
0 10-1 ml. Iungextract
given intranasally
3 Noobservable illness
7 Noobservable illness Peribronchial infiltration of small round cells
in rightmedian lunglobes
14 Noobservable illness Peribronchial lymphocytes, intrabronchial
granulocytes; consolidated of both right me-
dian lunglobes
30 Chattering Peribronchial plasma cells; consolidation of
right median and lower right lung lobes;
rhinitis, otitis; spleen enlargement
60 Chatteringandwastfng Depletion of lymphocytes perifollicularly in
spleen
90 Labored breathing Consolidation of right lung, emphysema of
followed bydeath left; depletion of lymphocytes in thymus cor-
tex hypergammaglobulinemia; spleen amyloid
Eight preselected miceofboth sexeswerekilled ateachtime.
and surrounded by cuffs of lymphocytes and plasma cells (Fig. 4). The
consolidated alveoli contained granulocytes and big foamy cells, prob-
ably macrophages (Fig. 5). The tympanic cavities contained exudate
with many polymorphonuclear granulocytes. Most spleens exhibited
anincreaseinweightofabout20%o.
After 60 days the mice were meager and chattering. Peribronchial
crescents of pyroninophilic plasma cells were prominent (Fig. 6). A
depletion of lymphocytes was apparent in the perifollicular zones of
the spleen. The cortex of the thymus was diminished. The tracheal
lymph nodes were enlarged. Mice surviving for 90 days in the terminal
222 EBBESEN Vol. 53, No. 2
phase suffered from consolidation of the entire right lung and gross
enlargement of the left lung due to emphysema (Fig. 7). In a few
pneunonic lungs invagination of peribronchiolar tissue into the bron-
chiolar lmen was found (Fig. 8). The invaginating cells in contact
with the lunilnal content looked like macrophages while more periph-
erically located cells resembled fibroblasts. The bronchiolar epithelium
was lacking on the invaginated tissue but was otherwise always present
in the bronchioli. The cortex of the thymus was smaller than that in
controls and contained greater numbers of large PAS-positive cells.
A few pneumonic mice also exhibited small thymus cysts (Fig. 9).
In the spleens of most of the pneumonic mice there were deposits of
amyloid (Fig. 10) which stained positive with PAS, thioflavine T, and
alkaline Congo red and showed birefringence and dicbroism. Reticulum
cells with PAS-positive cytoplasm were occasionally found adjacent to
spleen amyloid. Nearly all of 40 inoculated mice kept until spontaneous
death died with amyloidosis of the spleen, and one-fourth had hyper-
gammaglobulinemia (Fig. 11). No correlation was found between
hypergammaglobulinemia and spleen amyloidosis. Normal hematocrit
values (about 50) were obtained in all stages of the lung disease. The
number of circulating leukocytes and the percentage of granulocytes
were, however, often slightly elevated (to about 7 X 103, respectively
50%) inthe terminalstage.
Pernasal transfer of infectious material for 14 generations gradually
reduced the mean survival time from 103 to 58 days. The lesions were
the same in all transfer generations except for the disappearance of
spleenamyloidfromthetenthpassage andallfollowingpassages.
Attempts to isolate Mycoplom=n pulmonis from lung and in a few
cases also from exudate from the middle ears of 100 pneumonic and
20 nonpneumonic mice were kindly made by Dr. J. B. Nelson, The
Rockefeller University, New York;s Dr. L. Hayflick The Wistar Insti-
tute, Philadelphia,18 Dr. E. A. Freundt, Institute of Pathology and
Bacteriology, University of Aarhus, Denmark;19 and Dr. K Lind, The
State Semim Institute, Copenhagen. Mycoplsma pulmonis was never
isolated, and Dr. N. L. Somerson, Laboratory of Infectious Diseases,
National Institutes of Health, found no antibodies 2 to tis agent in
the mice. Dr. K Lind isolated Mycoplasma neurolyticum from the
lungs of 2 of 25 pneumonic and in none of 10 control mice. Mycoplamsin
neuroljticum, previously isolated from our BALB/c mice,2' was in-
oculated in amounts of 105 colony-forming units intranasally to 60
mice, intraperitoneally to 20, and subcutaneously to 10 mice. After 8
months the inoculum had failed to induce pneumonia. Staining for
August 1968 RESPIRATORY DISEASE 223
elementary bodies revealed a few intra- and extracellular Giemsa-posi-
tive Gram-negative bodies in imprints of a few pneumonic and normal
lungs.
The filtration experiments (Table 3) suggest a maxium particle
size 22 between 100 and 250 mu±. No lesions resulted from inoculation
ofinfectious materialinactivated byetherorheat (Table3).
Table 3. Filtration, Inactivation, and Neutralization of the Lung Agent Prior to Intra-
nasal Inoculation into BALB/c Mice; Influence on Occurrence of Pneumonia and of
Amyloid inthe Spleen
Mean Meangrade
Treatmt survival ofspleen
priortoinoculation Sex Pneumonic/Total (days) amyloid
Filtration
None M 10/10 123 1.3
None F 10/10 90 2.5
450 mni M 10/10 169 0.3
F 10/10 182 0.6
220 mp M 0/10 8mo. 1.3
F 8/8 226 1.3
100 MAi F 1/10 8 mo.
Etherinactivation M 0/30 8 mo. 0.3
56° C./30 min. F 0/30 8mo. 0
Neutralization
None F 10/10 72
Serum from normal BALB/c F 10/10 76
Serum from pneumonic BALB/c F 10/10 111
Serum from normal Swiss F 10/10 79
Serum from Swisswith gray
diseaseof lung F 10/10 86
Controls inoculated with filtred normal lungtissuedid notdevelop pneumonia.
The consolidated median lung lobes of 16 mice with terminal
pneumonia were examined for the presence of bacteria by cultivation
on blood agar, bromthymol blue lactose agar with crystal violet and in
serum broth.' In 9 of these mice no bacteria were detected. Pasteurella
pneumotropica was recovered from 3 mice, one of which also yielded
an anaerobic Streptococcus while another showed Ps. maltophilia in
addition. From each of the 4 remaining mice one of the following
bacteria were cultivated: Alcaligenes sp., Micrococcus sp., Pseudomonas
sp. andanonhemolytic Streptococcus.
Furthermore, bacteria were recovered from 2 of 6 untreated non-
' My than to Dr. H. Lautrop, The State Serum Institute, who performed this
investgation.
224 EBBESEN Vol.53, No.2
pneumomc controls. One yielded Eschericia coli and Hafnia alvei, an-
otheraMicrococcussp.
Hemagglutination occurred with mouse, rat, hamster, guinea pig,
rabbit, and hen red blood cells when the cells were incubated with a
suspension of pneumonic lungs up to dilutions of 1:16 or 1:64, whereas
suspensions ofnormallungs werewithout effect Serumfrompneumonic
mice and from rabbits immunized with pneumonic lungs failed to in-
hibit the agglutination reactions. Pneumonic hmg tissue had a con-
siderably higher phospholipid concentration than normal lung tissue as
judged from thin-layer chromatography of chloroform-methanol (2:1)
extacts.' Lung suspensions tested according to the technique for dem-
onstration of pneumonitis virus 3 did not cause any agglutination of
mouse red cells. No agglutination of red cells from pneumonic and nor-
mal mice was obtained with rabbit antiserum either in a direct or in an
indirect modified Coombs test. Cold agglutinins were not found. Single
radial diffusion and double diffusion in agar failed to demonstrate
precipitating antibodies tothelungagentinserum.
Incubation of lung extract with serum from pneumonic mice pnor
to intranasal inoculation resulted in a significant (p < 0.01) prolonga-
tion of the survival time, while serum from mice with grey lung disease
was withoutinfluence (Table3).
Discussion
The experimentally transferred disease imitated the spontaneous
pneumonia in our old BALB/c mice.5 The sequence of lesions ob-
served indicated a primary effect of the inoculated agent, leading to
a peribronchial accumulation of lymphocytes and, secondary to this,
intrabronchial plugging with granulocytes and sometimes also in-
vagination of tissue, resulting in consolidation oflung lobes andinvasion
of alveoli and peripheral blood with granulocytes. Both lung plasmo-
cytosis and hypergammaglobulinemia, when found, indicated a persist-
ent reaction to infective agents. Amyloid development is hardly caused
by the hypergammaglobulinemia" since in most mice the electro-
phoretic patterns were normal, but the amyloidosis could be due to the
PAS-positive cells found in the area of amyloid formation.25 Disappear-
ance of amyloidosis after some transfer generations was probably due
to the concomitant reduction in survival time." However, the possible
loss of an amyloid-inducing agent disfinct from the pneumotropic agent
inthetransfers couldnotbeexcluded.
*Thin-layer chromatography wa kidly pfled by Dr. S. E. Hansen in this
institute.
August 1968 RESPIRATORY DISEASE 225
The filtration experiments pointed to one or more large viruses, less
probably Mycoplsnas,2729 as the initial inective agent(s). Myco-
plasma pulmonis, which causes a very similar disease,"2 may be ex-
cluded owing to the repeated failures to isolate the agent and failures
to demonstrate specific antibodies, and the absence of coccobacilhiform
bodies 16 and possibly the absence of cold agglutinins.1 Mycoplasma
neurolyticum was sometimes found but apparently caused no lung
lesions. The agent causing grey lung disease'2 differs from ours since it
rarely leads to death, causes lung edema,50 and probably does not
induce neutralizing antibodies.3".32 Substantiating this viewwas thelack
of inhibition of our agent by serum from mice with grey lung disease.
The Nigg agent,,3 K virus," and the pneumonitis virus -3 all cause
more acute diseases, and pneuinonitis virus induces a characteristic
hemaglutination reaction which was not positive in our experiment.
Clinical and histologic findings in our mice are very similar to those
Nelson described for white mice infected with an agent which, from
filtration experiments, was considered to be a large virus, which causes
chronic murine pneumonia (enzootic bronchiectasis).2 The filterability
and the heat and ether sensitivity of the agent in our experiments would
fit with such a large virus as the etiologic agent, although Mycoplasma
would still be a possibility. Also pertinent to our agent is the virus re-
portedbyBatherandCushing.a
That more than one agent (vinrs) should be initiating the disease
is not probable, since the symptoms were identical in mice receiving
the different filtrates and since no lesions appeared in mice-receiving
ether- and heat-inactivated lung agent or receiving normal lung tissue.
Eperythrozoon coccoides, which might have caused the spleen enlarge-
ment,3W37 was not observed in our mice by microscopic eination of
blood from splenectomized animals.5 Most of the lesions observed in
the infected mice could be caused by the initial infective agent, since
the infectivity of lung material from terminal pneumonias proved the
persistent sojourn of the agent The granulocytic response and the isola-
tion of bacteria from some mice indicate that secondary invasion of
bacteria might perhaps be of importance for the late lesions. The
low incidence and the variety of the bacterial species found, however,
make it unlikely that the presence of such secondary invaders is a
necessaryconditionfordevelopmentofthelesions.
The negative outcome of the modified Coombs tests and of agar gel
diffusion experiments may be due to a lack of antigen attachment to
redbloodcells andtolowconcentrations ofappropriate antibodies.
The agglutination of red blood cells from several species with extract
226 EBBESEN Vol. 53, No. 2
from pneumonic lungs is probably nonspecific, as it was not influenced
by antiserums and as the lung extract seemed to contain much phos-
pholipid, whichmaycause uinspecific agglutinationu
It is thus to be concluded that our BALB/c mice are harboring the
agent (big virus? Mycoplasrna?) causing chronic murine pneumonia
and that neutralizing antibodies are present in serum of pneumonic
animals.
Summary
BALB/c mice inoculated intrnasally with material from lungs of old
BALB/c mice with spontaneous chronic respiratory syndrome died
with pneumonia after 2-6 months. An initial phase of peribronchial
accumulation of lymphocytes was followed by intrabronchial plugging
with granulocytes and by peribronchial infiltration of plasma cells. The
alveoli were filled with foamy macrophages and granulocytes. Amy-
loidosis of the spleen was found in most mice with terminal pneumonia
and one-fourth of them had hypergammaglobulinemia. Mycoplasma
pulmonis could not be grown from pneumonic lungs. The infective
agent passed ifiters with a pore size of 220 my but not 100 m,y and was
ether- and heat-sensitive; it could be partially neutralized with serum
from pneumonic mice but not with serum from mice with grey lung
disease. Suspensions of pneumonic lung tissue caused a probably non-
specific agglutination of red blood cells from mice, rats, guinea pigs,
hamsters, rabbits, and hens. The lung disease was considered a variant
ofchronicmurinepneumonia.
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Weare gratefulto Mr. BentBorgesenforthepreparationofthephotomicrographs.
Legends for Figures
Fig. 1. Right lower median lung lobe of young BALB/c mouse 7 days after intranasal
inoculation with lung extract from old untreated BALB/c mouse with chronic respira-
tory disease. Cells have accumulated around bronchioles which in most cases have
empty lumen. Periodic acid-Schiff (PAS) technique. X 35.
Fig 2. Greater magnification with cross section of bronchus from same lung lobe
as in Fig. 1. Most cells around bronchi are small lymphocytes. PAS technique. X 525.