LEUCOCYTOSIS ASSOCIATED WITH ACUTE
INFLAMMATION*
ANDERSON NETTLESHIPf
But little is known of the mechanism which produces leucocytosis in acute inflammatory conditions. From the area of local
damage some stimulus must travel to the bone marrow to cause its
heightened activity.
Understanding of this phenomenon may be gained by studying
acute inflammatory lesions in relation to the concurrent leucocytosis. In the present study small lesions were produced in the
skin of the rabbit. These were studied to see if necrosis at the
site of inflammation may be related to leucocytosis.
Experiments
The hemolytic streptococcus isolated by Gay1 was the inflammatory agent.
Rabbits with a normal blood picture were used. The usual blood techniques
were employed, smears being stained with Wright's stain. Lateral ear vein
blood was obtained, care being taken not to traumatize the ear as this produces
a leucocytosis (Schattenberg2). The blood picture was studied every two or
three hours during the first forty-eight hours, after that every twelve to twentyfour hours to termination of the experiment. Thirteen animals were injected
into the skin of the back with 0.1 cc. of a suspension of a 1:100 dilution of a
twenty-four hour broth culture of the virulent organisms. Six control animals
were injected with the same quantity of broth—they remained normal throughout the experiment. Total averages, in thousands of leucocytes, of the thirteen which received the virulent organisms are given in table 1. Total time is
two weeks.
Leucocytosis set in within an hour after injection. There was a rapid rise,
the leucocytes increased 100 per cent above normal by the eighth day, one animal at that time having a leucocytosis of 50,000, and leucocytosis of 25 to 35
thousand was commonly seen. This is not clearly brought out by the table
since each animal was at the height of its leucocytosis at a slightly different time
* Received for publication February 26th, 1938.
t Fellow in Medicine, The National Research Council. (This work was
begun in the Department of Pathology, Cornell Medical School.)
398
LEUCOCYTOSIS AND ACUTE INFLAMMATION
399
from the others. Peak heights persisted for eight to ten days. Though most
animals showed a return to normal at three weeks some had a moderate leucocytosis at that time. Leucocytosis is brought about by an increase in polymorphonuclear cells. Young forms make up part of the increase after the first
forty-eight hours.
In the skin, at the site of injection, a small reddened area, pointed with edema
and a tiny center of necrosis, was often visible between the eighth and twelfth
hour. This -pustule became rapidly larger during the first twenty-four hours,
the necrosis being as large as 10 by 20 millimeters at that time. Necrosis spread
less rapidly during the next twenty-four hours. Chart 1 shows a typical case,
animal number 1004. The gross appearance of the surface of the abscess can
not always be correlated, in time and amount of necrosis, accurately with the
leucocytosis since most of the necrosis is hidden beneath the skin surface. A
very accurate correlation with the micro-section is possible.
TABLE 1
TIME AFTER I N J E C TION
LEUCOCYTES
TIME AFTER I N J E C TION
thousands
Before
3 hrs.
6 hrs.
8 hrs.
12 hrs.
24 hrs.
36 hrs.
9.0
13.7
17.0
13.7
18.0
16.1
16.6
LEUCOCYTES
TIME AFTER INJECTION
thousands
48 hrs.
3 days
4th day
6th day
6th day
7th day
8th day
17.0
21.1
18.1
20.0
21.9
22.4
24.6
LEUCOCYTES
thousands
9th
10th
11th
12th
13th
14th
day
day
day
day
day
day
20.0
24.3
19.1
24.1
16.0
19.0
Histologic changes following injection. The microscopic changes will be described chiefly from the changes taking place in the leucocytes. Haemotoxylin
and eosin, Verhoeff van Gieson and eosin methylene blue stains were employed.
Two hours. At two hours the collagenous fibers are slightly spread apart by
edema. Scattered groups of polymorphonuclears are already present, most
abundant where there are masses of streptococci. Many leucocytes show cytoplasmolysis (fig. 1). (Cytoplasmolysis as used here and throughout designates
cells whose cytoplasm is "fading out", or the leucocyte which retains a bare
rim of cytoplasm, or simply naked nuclei.) Few leucocytes show nuclear damage. Blood stream leucocytosis is 25 per cent above normal.
Four hours. Edema is more advanced and the number of leucocytes somewhat greater (fig. 2). Leucocytosis is rising.
Eight hours. Edema is marked, leucocytes present in abundance, about five
times as many as at two hours. Their granules are often swollen and many
show marked cytoplasmolysis. Karyorrhexis is slight, only an occasional
400
ANDERSON
NETTLESHIP
nucleus shows more advanced lysis. Collagen fiber damage is slight, there
being no demonstrable connective tissue or elastic fiber damage. All these
structures are spread apart by edema but they retain their outlines and internal
structures. Blood stream leucocytosis continues to rise.
Twelve hours. Collagen fibers show distinct disintegration. There is a
small center of central necrosis, affecting in most part leucocytes. Leucocytoplasmolysis is extreme. Leucocyte nuclei, for the first time, show marked
karyorrhexis and karyolysis. Leucocytes are infiltrating in enormous numbers.
CHART I
HOURS 6
12
18
24
30
36
42
48
CHART 1. This chart illustrates the general blood stream leucocyte changes
and local changes following injection of 0.1 cc. of a 1-100 dilution of a 24 hour
broth culture of hemolytic streptococci. The heavy unbroken line represents
the leucocytosis and is typical for the group. The light broken line is the area
of visible necrosis (in the gross) given in square millimeters. The bottom pictures show the microscopic changes affecting the leucocytes in the abscess.
Note early necrosis—2 hours. At point A the collagen fibers first show necrosis—12 hours. At B the connective tissue and elastic fibers are beginning to
necrose. And at C, 24 hours, the epidermis and associated structures are beginning to necrose. Animal number 1004.
An occasional monocyte is seen about the outer edge of the lesion. Generalized
leucocytosis is 100 per cent or above.
Twenty-four hours. Though nuclear degeneration is more marked than at
twelve hours one is struck by the relative slightness of this in comparison to the
LEUCOCYTOSIS AND ACUTE
INFLAMMATION
401
T
4
'W-
*
• * *
#ft-#fc
4
L
j£**
^
*
%
rf
* * * * #
FIG. 1. Leucocytes in a two hour area of injury (0.1 cc. of a 1-100 dilution
of broth culture). The amount of cytoplasniolysis is striking, there being but
one intact cell visible in the field. X1700.
FIG. 2. Leucocytes in a four hour area. There are usually eight to ten such
groups to be found in the area of streptococcus injection at this time. Here,
too, cytoplasniolysis is noted in a large per cent of the cells. X1300.
402
ANDERSON
NETTLESHIP
amount of cytoplasmolysis which is extreme (approximately one-third of all
leucocytes, many of which are completely stripped of their cytoplasm). There
is marked swelling and fraying of the collagen fibers. Epidermis and associated structures stain poorly and are beginning to necrose. Leucocytosis remains over 100 per cent above normal.
Forty-eight hours. A piece of epidermis has sloughed out. Leucocytes are
packed in the abscess tightly. There are a few fair sized hemorrhages. Blood
stream leucocytosis is well: over 100 per cent above normal, in some animals
over 200 per cent.
Animals sacrificed at the second, fourth and tenth days showed marked hyperplasia of the bone marrow.
In summary: Streptococci introduced into the skin of the normal rabbit produced acute inflammation accompanied by an early,
marked, prolonged leucocytosis. Cytoplasmolysis of leucocytes
at the site of injection set in within two hours after injection and
became more marked during the first twenty-four hours of inflammation, at which time the most rapid blood stream increase in
leucocytes occurred. The cytoplasm of the leucocytes was the
only tissue which necrosed early. Necrosis of the other tissues
involved in the abscess did not occur to any extent until after
twenty-four hours.
DISCUSSION
Leucocytosis of acute inflammation differs from that termed
physiological leucocytosis (Garrey, W. E. and Bryan, W. R. 3 );
there is, therefore, no need to discuss physiological leucocytosis
here.
The mechanism which may be responsible for generalized leucocytosis with acute inflammation, investigated in this study is:
that some breakdown product from the action of the injuring
agent on peripheral tissue diffuses into the blood stream to cause
leucocytosis and bone marrow hyperplasia.
The sections studied show that the tissues at the abscess site
necrose at different times. In order of their necrosis were noted,
leucocytes, collagen fibers, connective tissue and elastic fibers,
epidermis and associated structures. The time of necrosis of
these various structures is readily related to the general leuco-
LEUCOCYTOSIS AND ACUTE INFLAMMATION
403
cytosis. In the gross we have observed that as necrosis advances
so advances leucocytosis. Microscopically, when the necrosis of
each peripheral tissue at the site of injection is analyzed in relation
to the time of leucocytosis, the one tissue whose damage comes
shortly before and which necroses parallel to the blood stream
leucocytosis is the white cells at the site of injection. Since
necrosis of the nuclear material of the leucocytes occurs relatively
late (18 to 24 hours) the observations point to the cytoplasm of
the leucocytes as containing the "factor," which travels in the
blood stream from the site of damage to produce myeloid hyperplasia and leucocytosis. Observations on human disease further
the point. Lobar pneumonia causes a high leucocytosis in its
early stages. The one tissue which shows marked necrosis early
is the leucocytes in the alveoli.
Since His and Zinsser4 first used leucocyte extracts in treatment
of infections a great deal of work has been done on this. Thompson (quoted by Alexander5) first found that the injection of leucocyte extract gave a marked leucocytosis. This has been widely
confirmed. The leucocytosis is primary and not preceded by a
leucopenia. Why leucocyte extract causes a leucocytosis is made
clear from the present work; the leucocyte extract acts on the
bone marrow just as do the breakdown products (may be the
same substance) from disintegrating leucocytes in the abscess.
A tremendous amount of work on protein and protein breakdown products, injections of all sorts, has been done. One of the
more recent of such studies is that of Doan et al.6 With such
injections there is a primary leucopenia followed by a leucocytosis.
The studies point to a primary leucocyte damage, the breakdown
products resulting from the primary damage causing the leucocytosis.
Through study of the abscesses it was observed that there was
a distinctly greater necrosis in the forty-eight hour abscess than
in the twenty-four hour one. The advancing edge of necrosis is
not increased proportionately. Because of this, and the fact
that the center of the abscess is necrotic at forty-eight hours, the
absorption of breakdown products is not so great. The leucocyte
curve is beginning to flatten out. Another factor accounting for
404
ANDERSON NETTLESHIP
the slowing down of acceleration may be that the bone marrow
becomes increasingly difficult to stimulate.
Since leucocytes go to pieces continually in the blood stream,
the mechanism for increased leucocyte production, suggested by
the present experiments, may simply be an accentuation of the
normal. The normal growth of bone marrow may be controlled
by the usual breakdown products from the continually disintegrating leucocytes.
I am grateful to Doctor E. L. Opie for his help during this work.
SUMMARY
1. Acute inflammation caused by the injection of hemolytic
streptococci intracutaneously is accompanied by a marked, prolonged leucocytosis. The leucocyte rise is made up of polymorphonuclear cells. There is marked bone marrow hyperplasia.
2. Necrosis of the cytoplasm of leucocytes infiltrating the
injured area is closely connected with generalized leucocytosis.
Necrosis of other damaged tissues is not directly temporally
related to the leucocytosis.
3. Substances released from the cytoplasm of necrosing leucocytes appear to diffuse into the blood stream and cause the leucocytosis and bone marrow hyperplasia which accompany acute
inflammation.
REFERENCES
(1) GAY, F. P., AND STONE, R. L.: Experimental streptococcus empyema.
J. Infect Dis., 26: 265-284, 1920.
(2) SCHATTENBERG, H. J., AND HARRIS, W. H.: Production of local leucocytosis
in the rabbit by mild provocative measures. Proc. Soc. Exp. Biol,
and Med., 29: 269-272, 1931.
(3) GARREY, W. E., AND BRYAN, W. R.: Variations in white blood cell counts.
Physiol. Rev., 15: 597-638, 1935.
(4) Hiss, P. H., AND ZINSSER, H.: Experimental and clinical studies on the
curative action of leucocyte extracts in infections. J. Med. Res..
19: 322-397, 1908.
(
5) ALEXANDER, D. M.: The use of leucocytic extract in infective processes.
British Med. J., 1: 355-357, 1911.
6) DOAN, C. A., ZERFAS, L. G., WARREN, S., AND AMES, 0 . : A study of
the
mechanism of nucleinate induced leucopenic and leucocytic states.
J. Exp. Med., 47: 403-435, 1928.