A Factor in Lungs of Anaphylactically Shocked Guinea Pigs

Which Can Induce Eosinophilia in Normal Animals

By MAX SAMTER, M.D., MARThA A. IOFOED, B.S., AND WILLIAM PIEPER, B.S.

T HE INCREASE in eosinophils in blood and tissue which develops after

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reinjection of a specific antigell in sensitized guinea pigs has been the object
of extensive study. The available evidence which we have summarized befor&
suggests that (1) neither antigen nor antibody alone produces eosinophilia; (2)
eOsi!lophilia is the result of an alitigen-antil)ody union ; (3) a maximum number
of circulating eosinophils is reached twelve hours after rei!ijection, preceded by
a transitory decrease; (4) in guinea pigs, tissue eosinophilia is most marked in
the penibronchial tissue; (5) although cO!isiderable variatio!i exists i!i the extent
of eoSi!iOphilia in different animals of the same species, the increase !i the tissue
roughly parallels that in the peripheral blood; and (6) eosinophilia in the pen-
bronchial tissue persists for days, and in some instances for weeks, while the
peripheral eosinophilia declines with forty-eight hours.
It. is noteworthy that many of the early workers in the field call attention to
the occurrence of eosinophils in peribronchial tissue-findings which are now
subject to rei!lterpretat.ion. Opie,2 for instance, reported t.hat. guinea pigs develop
eosinophilia of mesentery, lymph glands, and lungs aft.er ingestio!i of meat
infested with trichina. He attributed their presence t.o the “t.ra!lsmission of the
eml)ryonic parasite through these orga!is,” even though he was unable to demon-
strate the parasites in the penibronchial tissue. The more receiit. understanding
that antibodies are formed in cells of, or derived from, the reticuloe!idothelial
system3 and fixed in shock tissue in which antigen-antibody reactions take place4
conveys a new meaning to Opie’s findings.
It is uiot our i!ltentio!l t.o suggest that antigen-antibody uinoi represents the
only mechanism which can induce eosinophilia. Vaughn5 reported the develop-
meuit of a transitory eosinophilia which followed administration of a single in-

jectio!i of a soluble protei!i-free extract. of ascaris a!ld reached it.s maximum

twelve hours after injection. Vaugh!l assumed that the release of histamine was
instrumental in the production of eosi!lophilia-a point, of view which has 1)een
held by others6 but which needs further experimental work before it can be
substantiated.

From the I)epartment of Me(licine and the Allergy Unit of the University of Illinois
College of Medicine, Chicago, Ill.
Submitted March 17, 1953; accepted for publication Max’ 1, 1953.
This study was supporte(l in part by funds from the Asthmatic Childrens Aid, Chiicago,
Ill.
We are greatly mn(leh)te(l to I)r. Herbert C. Batso!i, Professor’ of Biostatistics, Depart-
ment of Public health, University of Illinois, for his recomme!idations regarding treatment.
and presentation of our data; to Dr. Leon L. Gershbein, for his constructive commenits on
thie arrangements of the eXl)eriments, for outlining plans for further ilidenitification of the
eosiniotactic substance, and for his generous criticisms of our w’ork and of the manuscript
i’eportinig it; to Drs. i\Iark H. Lepper and George G. Jackson, for their advice on reducing
infection in recipient guinea pigs; and to Mrs. Catheri!ie Bader and Miss I(oniai!ie Palicke
fo)r faithful and effective technical assistance.
1078
 SAMTER, KOFOED AND PIEPER 1079

The function of the eosiiiophil is not known. Attempts have been made to
correlate its prese!ice w’ith various phases of the antigen-antibody reaction.
(1 odlowski demonstrated that eosinophils carry ‘ ‘anaphylactogenic material’ ‘.7
Code believes that they remove histamine from the site of the antigen-antibody
unio!l.8 Vaughn, in fact, enlarged Code’s concept by stating that eosinophils
carry histamine or other toxic by-products of anaphylaxis from the bone marrow-

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to the tissues for detoxification.
As a result. of studies of the behavior of eosi!iophils in man and in experimental
animals, we concluded that the “eosinotactic stimulus” originates in the tissue
in which the antigen-antibody reaction takes place. We assumed that the
stimulus, whatever its nature, produces an eosinotactic substa!ice which causes
eosinophils to leave the vascular bed and to migrate into the penibronchial area
and that the same substance initiates the subsequent release of mature eosinophils
from the bone marrow into the circulating blood following their transitory

depletion immediately after the antigen-antibody reaction. Out’ present stu(iy

examines the possible existence of an eosinotactic suhsta!lce in lungs and other
tissues of normal, sensitized, and shocked guinea pigs. The basic experimental
pro(’edure consists of the transfer of tissue suspected of containing the substance
into the penitoneal cavity of normal recipients and the determination of the
number of circulating eosinophils before, alid twenty-four hours after, transfer.
Modifications of the same procedure were used to demonstrate the developmeuit
of the eosinot.actic factor within the same donor animal by removing pulmonary
tissue before and after anaphylactic shock and to compare the eosi!lotactic
potency of nonheated and heated tissues from t.he same source.

PROCEDURES

I. Transfer of Lungs from Normal and Sensitized Guinea Pigs to Normal

I? ecipients
Normal Animals-i to 1 Transfer

Liperunental procedure: Guinea pigs of either sex, bought at random, weighing

between 250 and 300 Gm., were kept on a standard diet of Hockland pellets
(vitamin C fortified), supplemented by carrots. After a period of observation
which varied from six to fourteen days, the animals were killed by intraperito!leal
i!ijection of 1 .5 ml. of a saturated solution of pentoharhital sodium. Immediately
after death the lungs were removed. Care was taken to prevent coiit.aminatioti,
although the relative immunity of guinea pigs t.o possible contaminants made us
abandon rigid sterile precautions. Slices of the lungs weighing 1)etween 450 and
500 mg. were then tra!isferred into the peritoneal cavity of normal recipient.
gui!lea pigs. With two exceptions, oniy recipients which had a pretransfer
absolute eosi!lophil count. below 100 cells per cu. mm. were used in the study.
Xonse!lsitized animals of this t.ype will i)e referred to as “normal” throughout
the report.

The surgical transfer was done under ether anesthesia. Anesthesia must be (heel) enough
to prevent abdominal reflexes, which are bounid to cause herniat.ion of the small intestines
through the abdominal wall and render tissue transfer difficult. After shaving, the abdom-
inal skin was incised in midline, muscles were separated, the peritoneum opened and held
1080 LUNG FACTOR IN EOSINOPIIILIA

by forceps on each side. After insertion of the tissue the incision was closed with thread in
separate layers; an average of three sutures was required. As a rule, the surgical procedure
was completed within four minutes after induction of anesthesia.
One slice of the lung used for transfer was fixed in formalin, embedded in paraffin, and
stained withi Giemsa stain for histologic examination. Eosinophil counts were taken of
the (honors immediately prior to the administration of pentobarbital sodium. The counts
were uniformly within normal limits, and the practice of taking eosinophil counts of the

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(honors was, therefore, aban(loned during the last third of the study. In retrospect this
appears unfortunate, since it would probably have eliminated an inconsistency in table 2,
where the recipients 20, 21, and 22, all of which received slices of the same presumably
!iormal (honor lung, showed it uniformly high peripheral eosinophil count after twenty-four
hours. We suspect that thie donor animal had parasites and should have been eliminated
from the experiment. The sections of the transferred lung showed an abundance of pen-
bronchial eosinophils. Eosinophil counts and total white blood cell counts of the recipients
were made before and twent v-four hours after transfer. Total white blood cell counts were
included to recognize superinnposed infection. The st.andards for thie technic of absolute
eosinophil counts have 1)een outlined in previous pubhicnttions.’

Results: The changes of the absolute eosi!iophil cou!its of normal recipie!its

which followed intrapenitoneal transfer of slices of lungs from normal donor
animals are recorded in the upper half of table 1. Each of the recipients in this

TABLE 1 .-Eosinophil (‘mints of Normal Recipients Before an(l Twenty-four Hours After
Transfe,’ of Lung Y’issue from Aormal and Sensitized Donors
(Absolute eosinophil count.s per cu. mm.)

Before transfer 24 hrs. after transfer

Source of tissue I ibof ___________________- _________ -

Mean Range Mean Range

Normal do!nors 16 16 0-61 63 . ()--233

Sensitized donors 16 22 0-56 44 0-89

group received lung tissue from a separate donor animal; thus, sixteen guinea
pigs had to l)e sacrificed to supply the tissues for the transfers. The average
absolute eosinophil couuit of sixteen recipie!its prior to tralisfer was twent.y-t.wo.
The average eosinophil count twenty-four hours after transfer was 44 cells per
cii. mm. The lowest pretransfer eosinophil count. in this group was zero; the
highest, 56 cells per CU. mm. The lowest and highest. eosinophil counts after
transfer were zero and 89, respectively. Individual counts did not appear to i)e
normally distributed. Accordingly, the range, rather thaui the sta!idard deviation
or stan(lard error, is considered the appropriate measure of dispersion.

Normal Animals-i to 3 Transfer

Experimental procedure: Four normal guinea pigs were sacrificed by inst rtpenito!iettl
injectio)n o)f 1.5 ml. of pentobarbital sodium. Immediately after death the lungs were re-

* Thie tables list only the arithmetic means. Due t.o the extreme vtniation 1)etweefl in-

divioluals, either a major (hffereflce between group averages or an inordinate number of in-
ohviolual observations would be required to establish the statistical significance of differ-
enices betweeni groups. The difference betwee!1 groups presented in tables 1 and 3 is so great
that statistical analysis is conisi(lered unnecessary. In the othier experiments (tables 4, 5,
6) the nuniber of individuals studied too limited to make statistical analysis prac-
ticable.
 SAMTER, KOFOED AND PIEPER 1081

moved, its previousl.v described. Of each of the donors slices of lung weighing between 450
an(1 500 mg. were thieni transferred into three separate recipients. Absolute eosinophil counts
of the recipients were obtained before, and twenty-four hours after, transfer. Sections of
each of the transferred lungs were prepared for histologic study.

Results: The changes of the absolute eosiiiophil counts of four groups of

normal recipients following the intrapenitoneal transfer of lung slices from

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normal donor animals are listed in table 2. The first group of recipients, all of
which received slices from donor animal 101, showed a marked increase in the
number of circulati!lg eosinophils twenty-f our hours after transfer. The remaining
three groups exhibited only insig!uficant. changes in t.he number of circulating
eosinophils.
TABLE 2.-Eosinophil Counts of Four Gi’oups of Normal Recipients Before and 7’u’enty-four
Hours After Transfer of Lung Tissue from Normal Donors
(Absolute eosinophil counts per cu. mm.)

Recipient
Source of tissue guinea pig Before 24 hours after
number

Normal donor * 101 20 80 585

21 42 336
22 6 433

Normal donor *102 24 19 3

25 39 28
26 S 44

Normal donor *103 27 6 11

29 14 6
30 46 19

Normal donor * 104 40 3 44

41 11 11
42 6 8

Sensitized Guinea Pigs

Experimental procedure: Guinea pigs of either sex, bought at random, weighiing between
250 a!id 3(X) Cm., were sensitized by the initraperit.oneal injection of 0.5 ml. of a 10 per cent
solutio!s of egg white i!s saline. Twenty-onse olas later they were killed by initraperitoneal
injections of 1.5 ml. of a saturated solutions of pentobarbital so(huni. Subseque!stly the ex-
perimental procedure was identical with that of the preceding group.

I? esults: The effects of the transfer of lung slices from sensitized but not
reinjected donors on the peripheral eosinophil count of normal recipients are
recorded in the lower half of table 1. The average absolute eosiisophil cOU!lt of
the recipients prior to transfer was 16. The average eosinophil count twenty-four
hours after transfer was 63. The lowest. pret.ransfer eosinophil count. i!l this
group was zero; the highest, 233 cells per cu. mm.

II. Transfer of Lungs from 1)onors Shocked with Egg White to Normal
Recipients
Expei’imental procedure: Guinea pigs of either sex, bought at random, weighing l)etsvecni
250 and 3(X) Cnsi., were sensitized by the i!)traperitoneal injection of .5 ml. of a 10 per cent
1082 LUNG FACTOR IN EOSINOPHILIA

solution of egg white in saline. Twenty-one days later they were killed by intracardial in-
jections of 0.5 cc. of the same solution. Only animals which died in acute anaphylactic shock
vithin three minutes after reinjection were allowed to serve as donors. Immediately after

death the lungs were removed, and slices weighing between 450 and 500 mg. were transferred
into the peritoneal cavity of normal receipients, as previously described. Absolute eosino-
1)hil counts of the recipients were obtained before, and twenty-four hours after, transfer.
Sections of the transferred material were prepared for histological study.

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Results: The changes of the absolute eosinophil counts of normal recipients
following intraperitoneal transfer of slices of lungs from animals shocked w’ith
egg white are recorded in table 3. Of ninety-one guinea pigs which received slices
of lung from donors sensitized and reinjected with egg white, twenty-three died
within twenty-four hours after surgery. The remaining animals appeared healthy
and showed no signs of discomfort. The average absolute eosinophil counts of
the recipients prior to transfer was 26. The average absolute eosinophil count
twenty-four hours after transfer was 269 cells pen cu. mm.
rfhe lowest pretransfer eosinophil count in this group was zero; the highest,
133 cells per cu. mm.

In accordance with reviously outlined experimental procedure, the pretransfer range

of the recipients should riot exceed 100 eosinophils per cu.mm. Two of the sixty-eight ani-

TAB I.E 3.-Eosinoph il (‘o u rts of Normal Recipients Before and Twenty-foii r Hou i’s After
Transfer of Lung Tissue from Donors Sensitized and Shocked with Egg White
(Absolute eosinophil counts per cu. mm.)

Before transfer 24 hours after transfer

Number of
-________
animals
--
____________________
Mean - Range
_________
Mean __ Range

68 26 0-155 269 0-1176

mals which form the basis for table 3, however, had pret.ransfer eosinophil counts of 100 and
155 cells per cu.mm. For this reason the animals should not have been included in the cx-
l)erirnent-their inclusion was caused by an experimental oversight.

rFhe lowest. eosinophil count after transfer was zero; the highest, 1176 cells

per cu. mm.

III. Transfer of Lungs from Guinea Pigs Sensitized and Rein,jected with Extract
of Ragweed Pollen Absorbed on Aluminum Cream into Normal Recipients
Experimental procedure: Ragweed extract containiing 2.1 mg. total nitrogen ler ml. was
mixed with equal parts of aluminium cream. The mixture was centrifuged. After cenitrifuga-
tions, nsitrogen was (letermined in the supernatant fluid. In the presence of nitrogen addi-
tional aluminium cream was added until the superniatant fluid became nitrogens-free. Of the
sediment, 0.25 ml. was then injected intramuscularly twice at a three day interval. Twenty-
one days after t.he second injection 0.5 ml. of a ragweed pollen extract containing 1.8 mg.
total nitrogen per ml. was injected into the right heart of the sensitized animals. After ansa-
phiy lactic shock the experimental procedure was identical with t.hat used in guinea pigs
sensitize(l and shocked with egg white.

Results: The changes in the number of circulating eosinophils of normal guinea

pigs subsequent to the intraperitoneal transfer of lung from donors seiisitized
and shocked with ragweed pollen extract are recorded in table 4. Of eight
 SAMTER, KOFOED AND PIEPER 1083

recipient guinea pigs, six w’ere alive and well twenty-four hours after surgery.
The meati eosii#{236}ophil coutit of the recipients before transfer was 24 cells per cu.
mm. ; twenty-four hours after transfer it was 329 cells pen cii. mm.

I V. Transfer of Sensitized and Shocked Lungs from the Same Donor into Normal
Recipients

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The evidence for the existeisce of an eosinotactic substance in the lungs of
shocked guinea pigs made us attempt to demonstrate its absence before aiid its
development during anaphylactic shock. This can be done because it is possible
t.o remove part of the lung or even a i’hole lung of sensitized donor animals prior
to reinjection of the specific antigen without apparent injury to its circulatory
system.

Experimental procedui’e: Guinea pigs were sensitize(l to egg white, its previously out linsed.
Twenty-one days later the animals were anesthetized by initraperitoneal insjection of a 3
per cent solutions of penstobarbital sodium in 10 per cent ethyl alcohol (0.5 ml. per l)oufld

TAB n.s 4 .-Eosinophil Counts of Normal Recipients Before and Twenty-four Hosi rs .lfter
Y’ransfer of Lung i’issue from Donors Sensitized and Shocked with an Inhalant A llerqen

(Absolute eosuiophil counts per cu. mm.)

Absolute eosinophils of recipient eer cu. mm.

Recil)ient guinea pig number . - . -- - -

Before 24 hours

402 44 288
403 11 333
404 (1 3SS
405 (1 488
406 55 122
407 33 355

Mean 24 329

of body weight). Thie chest of the aniesthetized animals yas shaved, cleaned, arid opened
on the righst side. A ligature was placed around the right hilus and part of the right lung
ss’as removed and transferred insto normal recil)ients. Immediately after removal 0.5 isil. of a
10 per cent solution of egg white was slowly injecte(l into the right heart under direct 01)-
servatioms. Shortly afterwards the breathing of the ansiniahs became labored. The volunie of
the left lung increased visibly, and its color chianged to a light pink. Within minutes the
animals (lied fl ansaphyhitctic shock. Upon death the left. lung was removed and slices were
t.ranssferred into the peritoneuni of normal recipients. Sections of both lungs were Prepared
for histologic study.

Results: The results are recorded in tables 3a and ob. lable Sb lists 1)0th
donors and recipients, so that the effect of t.he reinjection of egg white on the
eosinot.act.ic potency of lungs from each individual donor can be recognized.
In the group which received sensitized lung tissue, the average peripheral
eosinophil count before transfer was 23 aisd after transfer, 47. In the group which
received slices from lungs of the same domiors after injection of egg white, the
average eosinophil count of the recipients before transfer was 24 a mid after
transfer, 168 cells per cii. mm.
1084 LUNG FACTOR IN EOSINOPHILIA

TABLE 5.-Eosinophil Counts of Normal Recipients Before and Twenty-four Hours After
7’i’ansfer of Sensitized Lung Tissue and Shocked Lung Tissue from the Same Donor

(Absolute eosirsophil counts per cu. mm.)

Before transfer 24 hrs. after transfer

Source of tissue _________________________ __________________________

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Mean Range Mean Range

Sensitized Lung 16 23 0-55 47 0-89

Shocked Lung 16 24 0-100 168 11-411

Source of tissue Donor Before transfer 24 hrs. after transfer

Lung 636 sensitized 11 28

636 sensitized 0 44
636 shocked 22 139
636 shocked 22 161

Lung 637 sensitized 33 67

637 sensitize(l 11 44
637 shocked 0 144
637 shocked 22 278

Lung 638 sensitized 11 33

638 sensitized 44 44
638 shocked 22 167
638 shocked 11 122

Lung 643 seissitized 33 67

643 sensitize(l 11 67
643 shocked 22 200
643 shocked 67 377

Lung 644 sensitized 44 44

644 sensitized 11 89
644 shocked 0 322
644 shocked 11 411

Lung 642 sensitized 55 44

642 sensitized 11 44
642 shocked 1(X) 122
642 shocked 11 28

Lung 684 sensitized 22 11

684 sensitized 33 44
684 shocked 33 122
684 shocked 11 67

Lung 673 sensitized 28 28

673 sensitized 0 0
673 shocked 11 11
673 shocked 22 28
 SAMTER, KOFOED AND PIEPER 1085

V. Transfer of Tissue other than Lungs from Shocked Animals into Normal
Recipients
Experimental procedure: Female guinea pigs weighing 250 to 300 Gm. were sensitized to
egg white, as 1)reviously described. Twensty-one days later the itnirnals were sacrificed by
intracar(hial reinjection of the antigen. Immediately after death slices weighing from 450

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to 5(X) mug. of abdominal skin freed from hair, lungs, uterus, small intestine, liver, and gall
bladder were removed. The tissues were gently washed in saline and transferred into the
peritommeum of normal recipients.
Certain difficulties became apparent in l)relimiflary studies. A large number of
recipients (lied from strangulation caused by either strips of intestine or the horns of
the uterus or from acute peritonitis. Strangulation was minimized by cutting the
weighit units of intestinse (500 mg.) into small individual sections without mincing
and with only minimal loss of tissue fluid. Infection was suppressed by administering
streptornycins, orally, to the donor animals (0.5 Gm. twice a day, beginning twenty-four
hiours before reinjection) after it had been ascertained that streptomycin alone in
the (losage its which it w:ts given (hoes not appreciably alter the number of circulat-
ing eosinophils of normal guinea pigs.
As in the precething groups, sections of the transferred tissues were prepared for his-
tologic study.

TA B I.E 6 .-Eosinoph il (‘o,i rots of Normal Recipients Before and Twenty-four Hou ,s After
Trait sfer of la rioiis Tissues from A naph ylactically Shocked Guinea Pigs
(Absolute eosinophil counts per cu. mm.)

Before Transfer After Transfer

Transferred Tissue Oil -------- -- -.--- ------ -

Mean Range Mean Range

Uterus 6 29 11-56 43 11-67

Intestine 6 21 11-40 49 0-183
Liver 6 15 6-33 49 22-89
Gall Bladder 6 22 6-44 79 22-214
Lung 6 18 6-33 289 122-471
Skin ., 6 18 11-33 10 0-22

Results: rfhe results are recorded in tal)le 6. No death occurred among the
thirty-six guinea pigs which received transfers of various tissues from six donors
following a,aphylact.ic shock. Transfer of uterus increased the meami of t.he
circulating eosinophils of six recipients from 29 to 43 cells pen cu. mm.; of
intestines, from 21 to 41; of liver, from 15 t.o 49; of gall bladder, from 22 to 79;
of lunsgs, from 18 to 289; while the transfer of skin reduced t.he transfer of absolute
eosinophil counts in six recipients from a pretransfer level of 18 to 10 eosinophils
per cu. mm. twenty-four hours after transfer.

VI. Transfer of Nonheated am! Heated Lungs from the Same Shocked I)onor into
Normal Recipients
Experimental procedure: Guinea pigs senssitized to egg white were sacrificed twenty-one
days later b’ instracardial injections of the antigen, as previously (lescril)ed. Lungs were re-
moved immediately after anaphylactic death. One lung of eachi donor was placed ins a closed
container to) l)reveflt loss of fluid and kept. tt room temperature for 40 minutes. The other
lung, in an identicrtl contntiner, was incubated for 40 minutes at 58 C. At the end of 40 nun-
utes, sections w’eighing between 450 and 500 mg. of nonhieated and heated lung were trans-
ferred inito time abdominal cavity of normal recipients.
 1086 LUNG FACTOR IN EOSINOPIIILIA

I?esnlts: Results of the transfer of nonheat.ed and heated lungs from shocked
donors into normal recipients are recorded in table 7. The mean absolute eosino-
phil counts of tw’elve recipients vhich received iionheated lung tissue from two
shocked donors w’ere, 1)efore and 24 hours after transfer, 29 and 331 cells per
dl. mm., respectively. The low’est iisdividual eosinophil count. before transfer
was zero amid the highest, 61. The lowest individual eosinophil count after transfer

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i’as 25 and the highest, 1 176. The mean eosinophil counits of twelve recipients
which received heated lung tissue from the same donors were, before amid 24
hours after transfer, 31 amid 149 cells per cu. mm., respectively. The lowest
individual eosinophil count. in this group before tramisfer i’as zero amid the highest,
83. The lowest individual eosinophil count after transfer ‘as 19 and the highest,
722 cells per cu. mm.

TAB I.E 7 .-Eosinoph il (‘o u rots of Normal Recipients Before and Twenty-fooi r Hours
Aftet’ Trans,fer of Non.heated and Heated Lung Y’issue from
Anaphylactically Shocked Guinea Pigs
(Absolute eosinophil counts per cu. mm.)

Before transfer 24 hours after transfer

Source of tissue _____________ _____________ ______________________
Mean Range Mean Range

Noishmeated lung from

shocked doisor 12 29 0-67 331 25-1176

Heate(l lung front

shocked donor 12 31 0-83 149 . 17-722

DISCUSSION

Again, as in previous reports, one must stress the variability of peripheral

eosinophil counts pigs. Their in guinea
normal range is open to debate. On the
basis of published reports, the Committee on the Handbook of Biological Data
suggested a standard value of 400 cells per cu. mm. and a range from 200 to
1300 cells per tu. mm., but added t.hat that represented “approximate averages
in highly variable unweighted means and ranges from the literature.”10 The
figures appear high even for older animals which tend t.o develop a so-called
spontaneous eosinophilia”; they are too high for younger animals which are free
from parasitic infestation. In our experience the absolute eosinophil count of
healthy guinea pigs weighing from 250 to 300 Gm. was so consistently lower than
previous estimates that we have used an upper limit of 100 cells per cii. mm. as
a criterion for normality.
In recent years, considerable progress has been made in the investigation of
nonspecific regulations which control the level of eosinophils in the circulating
blood.’2 It has been shown t.hat pituitary and adrenocortical hormones mediate
the physiologic changes w’hich are observed, for instance, during a 24 hour cycle
and under stress.’3 In the light of this work, eosinophilia must be defined as au
increase in eosinophils w’hich exceeds the physiologic range of each species-a
distinction which facilitates the classification of our findings. It is true that the
transfer of lungs from normal and sensitized guinea pigs produces a significant
 SAMTER, KOFOED AND PIEPER 1087

iflclelLSe in circulating eosinophils of normal recipients. ‘The ilmeFease remains

w’ithin physiologic limits, ho’ever, ‘hile transfer of lung slices from shocked
donors pru(’es eosinophilia.
It appears that our study offers conclusive evidensce that aim eosinotactic
factor develops in the penibronchial tissue of guinea pigs subsequent to antigen-
antibody reactions of the anaphylactic type. The factor is absent in the lungs of

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seissitized guinea pigs but is found immediately after reitijection of the specific
antigen and persists at room temperature for at least forty minutes ---the maxi-
mum iimterval observed in our iimvestigatiomm. One might speculate, therefore, that
it develops concurrently w’ith the release of histamine (or H substance) from the
site of the aistigen-antihody reaction ; although one can only speculate that it
might 1)e part of the same immunologic mechanism. Vaughn arrived at the
conclusion that eosinophils carry histamine from the botie marrow to the tissues
where it. (fl be neutralized.’4 Our findings do not seem to be compatible with
\aughn’s concept. We now believe that the eosinot.actic factor originates in the
tissue and that eosinophils, for whichever reason, are attracted by it..
Thefact that eosinophilia (tt3 be engendered by the iistrapenitoiseal transfer
of shocked lungs us normal recipients demonstrates that the normal bone marrow
is capable of releasing mature eosinophils vithout prolonged incubation. The
incubation period of twenty-one days, which is required for formation and
optimal distribution of anaphylactic antibodies, is probably not associated with
an increased formatioti of eosinophils in the boiie marrow. Sustainsed eosinophilia,
on the ot.her hand, requires a persistent eosinot.actic stimulus.
The histologic examination of the transferred material fails to provide any
clues as to the nature of t.he eosinotactic substance. TTnless otherwise indicated,
sections of normal, sensitized and shocked lungs showed only an insignificant
number of eosinophils in the penibronchial tissue; in most. instances, lungs, as
w’ell as other tissues, were free from eosinophils, as is to l)e expected from animals
that (lie in ausaphylactic shock.
In analyzing the variations w’hich w’ere found in each group of recipient
guinea pigs it has been our impression that the increase in eosinophils in the
circulatinsg blood of the recipients following transfer w’as essentially a function
of the eosinotactic potency of the donor t.issue. Findings like the fairly uniform
increase in the circulating eosinophils of three recipients which had received
lung slices from the same donor (group 1, table 2) seem to favor this interpreta-
tioim. As the study progressed we became increasingly aware of the fact that. the
response of the recipient determines, at least to a certain extetit, the 24 hour
level of eosinophils following transfer of lung tissue from shocked donors. The
data make it evident that. event transfers from lungs which contain a (lemonst.rahle
quantity of eosinot.actic material fail to produce eosinophilia in at least some of
the recipients. The validity of our conclusions, therefore, rests w’ith the large
number of transfers which we have performed during the past two years.
Several possible reasons might account for the variation in the number of
circulating eosinophils in recipients after transfer of eosinotactic material. We
have previously mentioned that nonspecific regulations might depress an existing
eosiimophilia. The response of the animals to handling, for instance, might show
marked individual differences. On t.he other hand, the absorption of eosinotactic
1088 LUNG FACTOR IN EOSINOPIIILIA

material from the Peritolieal (a\’ity might take place at a different rate in
different recipieusts. In previous studies’5 we had investigated the fate of the
transferred material. The penitoneal cavity of the recipients was reopeised at
various intervals, amid traissferred tissues w’ere removed for histologic study.
Within hours after surgery the transferred slices were completely enveloped iii
omentum and often difficult to recover. Histologically, noise of the transfers

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show’ed eosinophilia: the histologic picture suggests a progressive autolysis which
appears to begin at the moment of transfer. Three weeks after traimsfer the only
remainder of the traussferred material is a small cyst-like mass of homogenous
material w’hich is, as a rule, attached to the posterior al)dominal vall. We are
inclined to assume that the eosinotactic factor is absorbed through the lymphatics
of the omentum. Interestingly enough, the lungs of recipients sacrificed at. the
height of the transitory eosinophilia which follovs int.rapenitoneal transfer of
shocked lung t issue generally showed marked peribronchial infiltration of
eosinophils.
Previous attempts to demonstrate an eosinotactic factor iii the serum of guinea
pigs after anaphylactic shock have failed. Thus Campbell was unable to produce
au increase iii circulating eosinophils in normal animals after the injection of
serum from guinea pigs which had developed eosinophilia sul)sequent to anaphy-
lactic shock.’6 In our ovui experience the injection of serum of patients with
peripheral eosinophil counts as high as (10,000 cells per cu. mm. failed to produce
a significant alteration of the peripheral eosinophil count of normal subjects.
Our findings that tissues of shocked donors other t.haii lung failed to induce
eosinophilia in normal recipients might be unexpected. Skimi tests and anaphy-
laxis in vitro (Schultz-1)al&7) leave imo doubt that. at least three of the t.issues
which we have transferred-skin, intestines, an(l uterus- carry antibodies. rf he

eosinotactic factor forms after antigen-antibody union. If tissues w’hich contain

antibody fail - -after aisaphylactic shock-to produce eosiusophilia in normal re-
cipients, one must assume either that their concentration of antibodies, com-
pared with lungs, is low’, or that they might not. participate in the anaphylactic
reaction of the guimsea pig, that, in other words, they might be antibody-con-
tabsing tissues but usot shock tissues. We favor the latter assumption in the light
of previous studies,’8 which have shown that the skin of shocked guinea pigs is
ordimmarily free from eosinophils, unless the specific auit.igen is introduced directly
into the skin by instradermal injection. The protective mechanism which dis-
tinguishes ant ibody-couitaining tissues from shock tissues is, of course, of great
theoret ic aisd practical interest.
The transfer of nonheated amid heated sections of lungs from the same shocked
donor has been undertaken in an attempt to elucidate t.he nature of the eosino-
tactic substance. The eosinotactic property of lungs is not reduced if they are
kept at 25 C. over a period of 40 minutes. Incubation at 58 C. for a period of
40 minutes seems to diminish the eosinotactic effect of t.he transferred material.
The experiments so far do not permit unequivocal conclusions.
Tim re-evaluating the immense literature on eosinophils in the light of our cx-
periments we have found many instances w’hich, like Opie’s report on the absence
of parasites iii the eosinophilic lungs of tnichina-infected guinea pigs, seemed
controversial at the time of their publication. Hajos,’9 for instance, senssitized
 SAMTER, KOFOED AND PIEPER 1089

guinea pigs amid re-exposed them to the specific aumtigen by inhalation. As a

result of the inhalation he recorded the development of pulmonary eosinophilia.
He attributed the eosinophilia to an “autonomic stimulus” vhich, initiated by
the bronchial mucous membrauie, amid relayed through autonomic chantiels to
the bone marrow, would cause a release of eosinophils and their return to the
site at which the stimulus had originated. In subsequent studies, how’ever, he

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readministered the antigen by injection amid called attention to the strange phe-
imomeuioum of a selective migration of eosinophils into the peribronchial tissue,
stating that he was uflaI)le to explain it. His findings are vell compatible w’ith
Our couicept which suggests the development of the eosinotactic factor at the
site of the aumt.igeli-alitil)Ody reaction.

SUMMARY AND CONCLUSIONS

I . Absolute eosinophil counts of normal guinea pigs were obtained before,

and tw’enty-four hours after, intrapenitoneal implantation of lung slices from
normal and seussitized guinea pigs, and of lung and other tissues-intestines,
uterus, gall bladder, liver, and skin-from guinea pigs w’hich died in anaphylactic
shock. Changes iii the number of circulating eosinophils of normal recipiemits
‘ere also determined after transfer of lung obtained from the same semmsitized
domior amsimal before and after reinjection of the specific antigen, and of lungs
from shocked animals incubated at 56 C. for 40 minutes.
2. Transfer of luuig tissue from normal amid semisitized (but. not reinjected)
guinea pigs into the penitomieal cavity of normal animals produces a sigmmificaimt.,
but limited, increase in the number of circulating eosinophils of the recipients.
3. rf1)f. of lung tissue of guinea pigs which died mi amiaphylactic shock imit.o
the perit.omieal cavity of normal recipiemits produces a marked transitory
eosinophilia.
4. Removal and transfer of one lung of sensitized guinea pigs, compare(I with
transfer of the other lumig after reinjectiomi of t.he specific antigemi demonstrates
that the eosinotactic factor develops immediately after anaphylactic shock.
Tramisfer of intestines, uterus, gall bladder, amid liver (compared w’it.h lung)
of shocked guinea pigs into the penitoneal cavity of normal animals produces a
small, but. significant, transitory increase in the number of circulating eosin-
ophils in the recipient.; transfer of skiim in a limited number of experiments appears
to depress the periphem’al eosinophil count of the recipiemmts.
6. Transfer of hmusg tissue from shocked donors incubated at 58 C. for 40 mum-
utes produces a less marked eosinophilia in miormal recipients than trauisfer of
lung tissue kept at room temperature for the same period.

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