- Email: [email protected]
Studies in experimental eosinophilia
STUDIES
IN EXPERIMENTAL
IV. Determinants Mortimer
Litt,
of Eosinophil M.D.,*
Boston,
EOSINOPHILIA
Localization Mass.
well established that tissue eosinophilia is not necessarily associated with blood eosinophilia.l, 2 The reasons for this dissociation are not clear, part,icularly if one considers that the bone marrow is the major, though not the only, site of cosinophil formation, and the blood stream is the vehicle by which these cells are transported to the tissues.2, 3 Most investigators agree that eosinophilotactic stimuli commonly arise at tissue sites (rather than in the blood stream), but the nature of the stimulus has been variously depicted. It has been held that this stimulus is (3.) an antigeq4 (2) a product of antigen-antibody union,“, (i (3) the antigen-antibody complex itself,7 and (4) histamine (one of the products of immune reactions) .’ This paper is concerned with the fact,ors which result in the localization of cosinophils at cxtravascular sites, and addresses itself to three subjects : (1) the identification of a transferable tissue factor which elicits eosinophilia, (2) the possibility that histamine is one of the mediators of the eosinophil response, and (3) the relationship between the site of challenge and the site of eosinophil infiltration.
I
T IS
MATERIALS
AND
METHODS
Guinea Pigs.-Female Hartley-strain guinea pigs, weighing 275 to 300 grams, were obtained from local breeders and fed Rockland rabbit pellets ad libit,um, supplemented by cabbage three times a week. Eosinophil Counts.-Peritoneal: The method of inducing peritoneal eosinophilia by repeated injections of foreign protein solutions, and the method of quantitating the eosinophil response by peritoneal lavage, have been described previously.” Blond: The ear veins were dilated by warm air from a hair dryer and nirk(d with a scalpel blade. Free-flowin, w blood was diluted 1:20 in a, 1 per cent l?rorn tbe Charming Laboratory, Boston City Hospital, and the Department of Bactcriand Immunology, Harvard Medical School, Boston, Mass. This work was supported by Grant DRG-588 from the Damon Runyon Memorial Fund for Cancer Research and Grant No. 506 from the Greater Boston Chanter of the Massachusetts Heart Association. Received for publication Sept. 26, 1962. *Research Fellow of the Helen Hay Whitney Foundation.
oloay
532
corrected white blood cell pipette with an acetone diluent,lO shaken for 30 seconds, and counted in an hcmocytomctcr. Counts were recorded to the nearest 10 cells.
Total Leukocyte Cozcnts.-The diluent was 2 per cent acetic acid. With the cascin-induced exudates, clotting was occasionally a problem. This was handled 1))~simply diluting the exudate at least 1 :I0 with saline before taking a sample for further dilution in the leukocyte-diluting pipette. Tissue Eosinophilia.-Skin: The hair over the dorsal aspect of the animals was rcmorcd by coarse and fine electric clippers, followed by an electric shaver. Six sites, one square centimeter in size and at least 2 cm. apart, were marked in ink, and intracutaneous injections through 27 gauge needles, were made at four of the sites. All six sites were subsequently removed, fixed in Bouin-Hollande,” embedded in paraffin, sectioned at +.L, and stained with Dominici.l* The total slumber of eosinophils in cross sections of the skin site (two sections, 4 mm. apart, were examined for each site) was determined by direct microscopic count. Footpads: Injections were made into both right footpads; the left footpads served as controls. The footpads were subscqnentlp removed and processed in the same way as the skin. Chenzicals.-Histamine diphosphate* was dissolved in 0.85 per cent saline. All amounts are expressed as free base. (In order to facilitate comparisons when citations are made of the literature, all doses of histamine have been con\-erted, when necessary, to the amount of histamine base; the conversion factors were 0.6 for histamine dihydrochloridc and 0.36 for histamine phosphate.) Horse serum was obtained from the Commonwealth of Massachusetts Department of Public IIealth Antitoxin and Vaccine Ilaboratory, Boston, Massachusetts. Hemocyanin was prepared as described previously.” Tprodc’s solution had a modified formula, described previously.13 Prepnmtion of Guinea 1%/s for Peritoneal Studies.-The method employed has been described in detail previously. I3 Essentially, each guinea pig was given weekly intraperitoncal injections of either 1 ml. horse serum or 0.5 ml. of 0.5 per cent hemocyanin for about 4 months, at which time challenge with antigen regularly produced peritoneal eosinophilia. When the eosinophil response to the last preparatory injection of antigen had ceased, the ant,igen was administered 1,~ a new route: intravenously in a le,e vein or subcutaneously in the ventral abdomen, either alone or accompanied by a simultaneous intraperitoneal injection of 8 ml. of sterile I:! per cent sodium cascinatc:t Some animals were tested with intrapcritoneal cascin alone. Whcncver a particular animal scrvcd as subject for testing more than one type of material, an int,raperitoncal challenge with antigen was interposed to verify that the animal was still in a state of responsiveness to the usual cffectirc stimulus (intraperitoneal antigen). *Pfanstiehl f~utritional
Laboratories. Biochcmirals
Inc., XVaukegan, Corp., Cleveland,
Illinois. Ohio.
534
November-December,
J. Allergy 1962
Those guinea pigs which were inoculated exclusively by the intracutaneous I-onte wwived 0.1 ml. of 0.25 per ccrrt h(~rrrocy:rrrin in each ventral abdominal quadrant each week. E’r~pnmtion cud Alssc~y of fCc:xtmcts of Peritoneal Lining.-Ten sensitized guinea pigs serrcd as tissue donors. Sftrr it, had been ascertained that, intraperit,oneal hemocyanin evoked a peritoneal eosinophil response, the guinea pigs wwc sacrificed (7 to 1-l days after the last injection). The peritoneal lining was removed, rinsed in two 10 ml. changes of Tyrodc’s solution, cut into 1 cm. strips and shaken in Tyrode’s solution for 4 hour~s at 37’ C. The strips were removed by filtration through cotton gauze and the liquid portion was centrifuged at 10,000 r.p.m. in a X3-1 Scrvall ecntrit’ugo. One hundred and fift,y milliliters of clear supernatant was frozc:n until USA. The extract, was tested in vitro for antibotl~ and in viva for eosinophilotactic activit,y both before and after absorption of the antibody by hernocyanin. The in yi1.o tests wer~c conducted 1)~ mixin, cr 7.5 ml. of extract with 0.5 ml. of 0.5 per cent hemocyanin, incubating for 30 min. at 37O C., and injecting the mixture into the peritoneal cavity of animals preparcd with horse serum (which is non-cross-reacting in this system) : total peritorrc~al eosinophil counts wcrc ma&~ 2-l hours following injcctiori.” The tests for antibody consisted of interfacial precipitation with antigctn in capillary tubes and double diffusion in agal’ gel against antigen; in the Iattc>r cast’, two distinct. bands formed after 21 hours. Srvcnty-t\vo millilitc~rs of the ext,ract was absorbed by two sequential additions of 1 ml. of 11 per cent heniocyanin. :jftcr ~a(~11addition, the solution was incubatetl for :! hours at 3’ (1.> and I)Iacctl at 3’ (‘. for 7 days. The resultant precipitate was rcmoyc>cl by cent rifugation. I~‘ollowing the sc~ontl portion of antigen, tests of suI)crnatarrt showed antigen in CSWSS.Both antigen and any soIublo complexes whic~lr nright Iiavc becir pr(lsclnt \v~‘r(l ~rtro~c~l by ccnt,riThe fugation at 37,000 r.p.m. Jc)r 2 hours irr a Sl)inco JIotl~l T1ult~accntrifugc. wp~~rnstnnt contain& neither amtig(ln nor+ arrtibotlp. as tlctcrwinetl by ring test ant1 diffusion in agar gel (2 weeks).
ophiZia.-It has been rcportcd”; that an extract of sensitized peritoneal lining, in association with the sensitizing ant,igrn, could induce peritoneal eosinophilia ; the active material in the extract was not identified. Table I shows that such an extract, which was active (when associated with antigen) in producing peritoneal eosinophilia, lost this activity when its antibody content was removed by specific absorption with antigen. The unabsorbed extract produced eosinophilia in 7 of 10 recipients, whereas the absorbed extract was inactive in all of 7 recipients. ‘1. The
Possible
Role
of
Nistcrmi~~~
as
(I XecLiintor
of
the
Eosinophil
Ir’es2)onsc,.-~-Tlrtt striking parallelism lwtwwn the c~ircumstances which result in lristarninc~ release and those which result in cosinophilia provide ample reason to consider histamine as a possible mediator of eosinophilia. If histamine does
Volume 33 Number 6
STUDIES
IN
EXPERTMENTAL
EOSINOPHILIA.
1V
535
TABLE J. EOSINOPJIILOTACTIC ACTIVITY OF EXTRACTS OF PERITONEAL LINING FROM GUINEA PIGS SENSITIZED WITH HEMOCYANIN GUINEA PIG I 11
Response
to
sensitizing
12 I3 Unabsorbed 8” 3
2 to
6
protein 22
Response
I4 Extract
extract
21
26
23
15
I6
I7
I8
6 4 3 6 1 ml. horse serum 39 12 17 25
I9110 8
6
6
22
40
21
4
0
5
2
3 4 4 1 0 1 5 7.5 ml. extract + 0.5 ml. - 0.5% hemocyanin 10 10 10 6 18 14 12
GUINEA PIG 11 1 12 1 13 j 14 I 15 1 16 1 17 1 18 2 Response
to
sensitizing
protein 18 5
Response
to
Absorbed 3
extract 7
13
Extract 6 3 5 6 3 1 ml. horse serum 18 18 14 43 20
5 2 4 2 4 7.5 ml. extract + 0.5 ml. - 0.5% 4 3 3 2 6
1 13
6 3 hemocyanin 2 1
*Each set of ~‘esponses shows the total number of eosinophils in millions, which wel‘e present in the peritoneal cavity immediaiely before and 24 hours following each injection. The recipients had been prepared by 4 months of weekly intraperitoneal injections of 1.0 ml. horse serum.
mediate eosinophilia it should be possible to bypass the long induction period which is typical of the usual method for inducing experimental eosinophilia. Instead of requiring weeks of repeated injections of foreign protein, eosinophilia should occur soon after the administration of histamine. A series of experiments was carried out in which the consequences of histamine administration were examined at each of four sites in guinea pigs. In the peritoneal cavity: Histamine was injected into the peritoneal cavity of guinea pigs and the total peritoneal eosinophil count was determined 24 hours later. This interval was chosen because, when sensitized guinea pigs are challenged by antigen, the peak accumulation of eosinophils occurs in about 24 hours.” The doses of histamine were 0.1, 1.0, 10, and 100 pg ; 4 guinea pigs were injected with each dose. No cosinophilia resulted in any case. The mean number of eosinophils found was 1.5 million and the range was 0 to 3 million. This is comparable to t,he number, 2.7 & 1.0 million, found in normal uninjected guinea pigs. o Since the duration of action of parenterally administered histamine is relatively short, it was possible that by 24 hours, when the assay was performed, the response was already over. In addition, the response might require contact with histamine that is more prolonged than that provided by a single dose. Accordingly, another group of 16 animals was given the same doses of histamine, but in a course of injections; the dose was repeated every 8 hours for a total of three doses, and the assay was made 8 hours after the last injection. Again, no eosinophilia resulted. This time the average cosinophil count was 0.25 million, and the range was 0 to 1 million. In skin: In order to determine whether hist,amine could attract eosinophils into skin, histamine was injected in the posterolateral wall of a group of 30
T11’1’ ‘1’
536 TABLE
II.
GUINEA
BLOOD
PIG
EOSIKOPHILIA*
1
IN
Qurmn
PIGS Fo~r,owxm HISTAMINE IiOlJItS
i-0
J& 0
10 10
November--l)ecemher,
TNTIMPER~TONEAL ALTER
10 0 10 10 0 10 10 10 10 30 0
10 40 90 10 30 10 80 IO 90 0 0
:: 20 21
10 10 10 30 0
50 40 40 50 10 10
80 50 40 :iO 50 10 0 30 0 0
100 50 0 30 10 0 0 30 0 0
22 23 24 25 26 27 28 29 30 31
30 CO 90 30 40 60 0 IO 90 50
ii 60 10 0 50 0 10 30 10
50 130 40 30 40 30 0 40 130 40
50 90 40 180 100 50 0 0
30 IO 40 50 30 0 30 50 40 40
40 40 50 50 20 50 40 100 20 20
150 30 40 50 10 60 40
150 10
2
0
4 5 G
0 0 0 0
180 100 50 30 0 50 30 30 I0
7 8 9 IO
11 12
10 50 30 80 0
13 14 15
1 1 1
1s 17
5 5 5
5 5 5 5 5 5 5 10
32 33 34 35 36 37 38 39 40 41
10 10 10 10 10 10 10 10 10 50 50 50
42 43 44 45 46 47 48 411 50 51
50 30 50 50 50 50 50
100 100 100 100
52 53 54 55 56 57 58 *lLkminophils
lili 40 60
100 100 100 per
cubic
10 60 10 30 0 JO 190 40 0 90 1;:
100 millimeter.
30
180 10 60
190 10 10
10 310 40 0
IO 280
10 30 150 100 0
100
80 10
::
ii 60 0 50 150 190 30 0 450 430 40
50
40 50 130 2x0 X0
2 150 300 450 '30
250 80 50 (I 60 280
MO 100 50 CI 490 250
190 130 100 30 930 50 640
IXJECTIOS
OF
IN,JIXTION
I_ 30 0 90 30 30 10 10 10 10 30 0
J. Allerpr 1962
30 100 80 30 fin 160 30 90 80 0 0 140 10 100 130 3-n 40 10 40 6: zi 10 40 50 30 0 60 90 50
Z-1 10 30 30 80
/
4x 10 90 ii
10 10
0 30
1.4: 1 A0 0 0
IO 60 180 40 0
10 30 30 50 50 50
50 40 0 360 40 50
3: 50 0
10 90 30 RO
140 40 140 40 ii
100 60 40 80 30 180 10
1: 10 60
1:: 130
480 190 60
0 30 100 50 50 60 110 50 30
30 50 40 80 0 50 40 40 10
100 430 340 30 160 140 210 350 530 240
10 130 80 10 30 100 40 100 160 80
130 10 50 80 10 140 80 50 50 10
130 60 130 80 1,010 530 490
260 '0 ;'O 30 350 200 450
50 80 40 10 60 40 40
630 50 0 50
10 250 130
Volume 33 Number 6
STUDlES
IN
EXPERrMENTXL
TABLE
GIJINEA
PIG
HISTAMINE (P!T)
EOSINOPHILIA.
II.
Cont’d
2
/
HOURS 0
59
100
60
:: 6% 63 64
100 100 100 100
.50 10 60 50 0
)
340 60 30 80 60 0
4
AFTER j
6
130
700
30 10 110 40 50
100 "00 30 40
537
IV
INJECTION 112
/
1,340 30 50 50 140 190
24 560 600 50 40 40
/
4X IO 10 30 10 110 0
guinea pigs. Each animal received four different doses-O.& 1.0, 10, and 100 pg-each at a different skin site, and groups of 5 of these similarly treated animals were sacrificed at 1, 2, 4, 8, 12, and 24 hours, respectively. The skin sit,es were removed for histologic cxsminabion. Eosinophils were found in only three of the 180 sites (120 experimental, 60 control) and numbered only 1, 4, and 2 per section (the latter was an uninjected site). In footpads: Similar experiments were carried out, in which the injections were made into footpads. Groups of 5 guinea pigs each were injected in both right footpads and all 4 footpads were subsequently removed. The first group of animals received one microgram of histamine at each injection site, the second group received 10 pg, and the third, 100 pg. The sites were examined 24 hours following injection and no eosinophils were found in any of the sections. The experiment was repeated, except that, the specimens were removed 4 hours after the injections. In nine of the thirty injected sites, a few neutrophils were found along with between one and eight eosinophils. In the hlootl stwcrnt: In studying the effects on the blood eosinophil count of an intraperitoneal injection of histamine, the experiments of Vaughn8 were repeated, except that t,he dose of histamine covered a loo-fold range. A group of 11 noninjected guinea pigs served as controls and they demonstrate (Table II) the degree of fluctuation which may be encountered in the course of collecting blood samples over a 48 hour period: the average starting count was 47 per cubic millimeter, while individual counts during the ensuing 2 days ranged as high as 180 per cubic millimeter.* Another group, consisting of 53 guinea pigs, with an average zero count of 41 per cubic millimeter, was injected with various doses of histamine, ranging from 1 to 100 pg. There was no apparent response to either the 1 pg or 5 pg dose, except for a single isolated high value (360 per cubic millimeter) at 48 hours in the case of the 1 ,LL~dose. With higher doses, there were definite responses. Four of the 10 guinea pigs which received the 10 pg dose had, at one time or another, an clcvated count, ranging from 190 to 630 per cubic millimet,er. A fivefold greater dose, 50 pg, resulted in elevated counts in 6 of the 10 recipients; thcsc counts ranged from 230 to 430 per cubic millimeter. The highest, dose, 100 pg, resulted in responses in 7 *The group means at any particular time and dose exhibit very large standard deviations. This reflects the fact that there at-e two classes of guinea pigs: responders and nonresponders. If the results are pooled one would conclude that there are no statistically valid effects. Yet it is grossly evident that some injected animals attain abnormally high values. Therefore. the results are prfxente~l as obtained in individual animals. A count greater than 180/mm.3 was considered abnormal. It is the highest value obtained in the control group and is about thrw standard deviations above the nlean of all the control counts. The normal values cited in the literature COVVI a wide range and the importance of working with young animals and using internal standards has been emphasized.5
53x
November-December.
J. Allergy 1962
of 70 recipient,s, and the level of response was higher; 4 animals had at least one count in excess of 500 per cubic millimeter, two of which exceeded 1,000 per cubic millimeter. In general, the initial response was noted as early as 2 hours, with the peak responses coming at 6 or 12 hours. All reaction had ceased (with the one possible exception already noted) by 48 hours. 3. The Relationship Between the Bite of Challenge and the Site of Eosinophi1 Infiltration.-Eosinophilia has been induced experimentally in a wide variety of tissues : conjunctivae, peritoneal cavit,y, skin, blood stream, muscle, lung, intestine. 2, 9, 15, 24, 31, 32, 33 While the sensitizing injections may take place at various loci, the predominant eosinophilia takes place at the site of deposition of the challenging dose of antigen. It is not clear whether the specific cosinophilotactic stimuli are generated at the challenge site or whether nonspecific inflammatory stimuli result in local cell accumulations during a generalized systemic reaction. The following experiments were undertaken to explore this question. In the peritoneal cavity, eosinophilia will result from repeated injection of foreign protein.9 Following a course of weekly intracutaneous injections into the ventral abdominal wall, the same result ensued. By the fourth injection of hemocyanin (0.5 ml. of 0.5 per cent solution), 9 of 16 guinea pigs had developed peritoneal eosinophilia; the average value was 19 million and the range was 13 to 27 million. In guinea pigs which have been sensitized by the intraperitoneal route, a challenging inject,ion of antigen results in peritoneal cosinophilia when it is administered intraperitoneally, but not when it is given intravenously (Table III). Nonspecific inflammatory factors, associated with the intraperitoneal injection might have accounted for the localization of eosinophils. Such animals have increased stores of eosinophils in their bone marrow,9 which might be “swept out” as part of a general leukocytic response. However, this is not the case, since the introduction of casein into the peritoneal cavity, while it evoked a striking leukocytic response in the peritoneal cavity (944 million to 2.4 billion total leukocytes), did not evoke an eosinophil response: less than 1 per cent of the leukocytes were eosinophils and their total number (0 to 9 million) was very small compared to t,he response which occurs upon injection of the specific sensitizing antigen. However, in the absence of antigen, few eosinophils might be released, making few available to be localized nonspecifically. Therefore, the sensitizing antigen was reintroduced, but by the intravenous route; thus it could act in the blood stream, or at any site served by the blood stream. Simultaneously, rasein was introduced into the peritoneal cavity, in order to encourage the localization of large numbers of leukocytes. The resultant peritoneal exudate contained large numbers of leukocytes ranging from 30 million to 6.8 billion. Yet, in spite of these apparently farorablc circumstances, few of the cells in the exudate were eosinophils. The total number of eosinophils did not approach what was found in the same animals upon peritoneal cha.llenge, and, in fact, the numbers were virtually what one finds in a normal, unstimulated animal.” Thcrc were two exceptions : guinea pig No. 9 had about as many cosinophils in tht> esudate as were found following intraperitoneal ant,igenic stimulation, but only
STUD1ES
L’olume ii Number 6
1N EXPERIMENTAL
EOSINOPHILIA.
IV
539
0.2 per cent of the total cells were eosinophils, whereas following intxapcritoneal antigenie stimulation, 25 per cent were eosinophils. In the case of guinea pig No. 8, the only one which showed a substantial nnmber of eosinophils (57 million), only 1 per cent of the total cells were eosinophils, as against about a 45 per cent eosinophilia followin g intrapcritoneal challmge with antigen (in the absence of casein). In a single instance, the antigen was administered subcutaneously, along with a simultaneous intraperitoneal iqjection of casein. Here too, there was no significant eosinophil response. TABLE
TII.
E:FFECT
OF THE ROUTE PERITONEAL
OF ANTIGENIC ~OSINOPFIlI~IA
HTIM~~ULATIO~~~ ON THE IN GUINEA PIGS GIJIiYEh
1.1’. ROI’TE
(‘ASEIN
OCWRREK~E
OF
PIG
1~2/3/4~516,7~819
IT.”
30t
17
20
15
16
45
40
30
10
I.V."
76 3
120 4
50 1
45
50
105
90 1
75
40
--_ 15
26
210 4
9
0
t I .v. is. c . *
0
t t
8 114 -
12
30 -
-
340
9 _920
-
-
4 1,650
944 2,400 2,200 4 8 57 12 ___-~ 200 6,800 6,000 6,000 6 900
Guinea pigs l-6 were prepared hemocyanin. The preparatory and Guinea pigs 7-S were prepared horse serum. The preparatory and Determinations were made 24 *I.P. = intraperitoneal. I.V. = tNumerator denotes millions Denominator denotes millions
by 4 months of weekly intraperitoneal challenging dose was 0.5 ml. of 0.5’jb solution. by 4 months of weekly intraperitoneal challenging dose was 1 ml. of horse serum. hours after the challenging injection. intravenous. S.C. = subcutaneous. of peritoneal eosinophils . of peritoneal leukocytes
injections
of
injections
of
DISCUSSION
Active sensitization of guinea pigs by the intraperitoneal route resulted in an accumulation of eosinophils in the peritoneal cavity and the localization of antibody in the lining of the cavity. This antibody, when extracted, complcxcd with antigen, and passively transferred int,o the peritoneal cavity of other guinea pigs, resulted in the rapid mobilization of eosinophils in the peritoneal cavity of the recipients. The activity of the extracts reported here and previouslyl” is related to the antibody content of the extracts, since removal of the antibody by precipitation with specific antigen abolished the activity. A similar activity in the serum of sensitized animals had previously been attributed likewise to its antibody content.’ While this earlier finding, that immune complexes containing serum antibody induced eosinophilia, suggested t,hat such complexes might mcdiatc the emigration of cosinophils from their bone marrow stores into the blood stream, it could not account for the more prominent localization at the site of introduction of the antigen. The current findings indicate that the mechanism of experimental tissue eosinophilia, as commonly
540
LITT
November-December,
J. Allerry 1962
produced by repeated injections of foreign protein, includes two stages: the preparatory injections of antigen result in the production of antibody and its localization at tissue sites, while the challenging injection of antigen combines with tissue-stored antibody. The resultant immune complex attracts the eosinophils. It seems possible that the activity which Samter, Kofoed, and Pieper” found in the lungs of guinea pigs who were anaphylactically shocked was also related to an immune complex. While Cohen, Kantor, and Gatto” found that the introduction of antigen and antibody at separate sites in the footpads of rabbits resulted in eosinophilia in the draining lymph node, they found t,hat irnmune complexes formed in vitro wcrc inactive in this respect. On the basis of these observations, they concluded that eosinophilia is not mediated by t,he immune complex itself, but rather by sonic product of the interaction of antigen, antibody, and some tissue component. Because of the differences in species, antigen, and route, the above experiments arc not strictly comparable with ours. Histamine is probablv not a major mc~tliator of the eosinophil response. It did not provoke an accumulation of cosinophils at any of the cxtravascular sites at which it was introduced. The results of the peritoneal studies are in accord with those of I’anzcnhagen and Spcirs,17I4 ~vho reported that 18 pg of histamine caused no eosinopbilia in the peritoneal (aa\-it,>-of mice, while 72 pg caused only a slight increasc.l” Relatively little histamine is stored in the skin of guinea pigs* and only insignificant amounts of histamine are released during allergic skin reactions,l’ yet even if all of the histamine were activated, it could not account for eosinophilia-one hundred t,imes t,he total cndogenous amount failed to produce any eosinophil responscl. In the horse, on the other hand, the intracutaneous injection of hist,amine does result in a local accumulation of eosinophils, but the response is substantial only with large doses of histamine. Thus, 900 ,~g,l~ 90 pg,l’ and 720 ,~g~” of histamine create foci in which the average coneentrat)ion of eosinphils is about 20 per high-power microscopic field. Ilower doses result in a mild eosinophilia: with 36 ,.~g$,the concentration averaged 6 to 7 per high power field,“l and with 3.6 pg, the concentration averaged 4 to 5 per high-power field.“’ AS Archer points out, presumably no local concentration of histamine approaching the level of 72 pg could occur in life, and it is questionable whether lower amounts occur. Yet even ~creral hundred micrograms of histamine does not reproduce the degree of eosinophilia which occurs in allergic humans and in experimental animals.““, 24 The results of the blood studies confirm those of Vaughn83 25; an eosinophil response occurred in the blood stream of some guinea pigs given large doses of histamine, but these were amounts of histamine which are not likely to bc encountered ill vivo. The cffccti\c doses, 10 pg and gr’cater, are larger than the amounts which arc activated by an alltbrgic event. Hven lethal anapbylaxis releases only a few micrograms of histamine: about ‘1 pg arc rclr:~ed from the *A footpa< contains about ~mrl), and 1 cmp of dorsal skin X 240 mg. per site).
1.2 pi’ of histamine (an injection site)
(i.7 gg contains
per gram’6 X 3 60 mg. per footabout 0.7 pg (2.9 86 per gra~rlx
Volume 3 3 Number b
STUDIER
IN
EXPERTMENTAL
EOSINOPI~ILIA.
IV
541
lung.2G Contrary results have been reported when somewhat different experimental designs were used. Campbcll27 found no rise in the cosinphil count after hc administered 500 ,~g of histamine intral)c~ritoncallv~all~ thrice daily for 3 days and sampled the blood every 12 hours. SanltW” also found that histamine (75 pg intracardially) did not induce eosinophilia. in normal animals 20 hours after injection; however, in animals sensitized to horse serum t,hc injection of histaThis parallels the situation in human mine alone did result in eosinophilia. beings in whom histamine alone provokes little eosinophilia unless the subject is allergic, in which case a significant cosinophilia occurs in the wheal produced by an intracutaneous injection of histamine.‘g, 3n Thus, while histamine can cause minor accumulations of eosinophils in the skin of horses18-22 and substantial eosinophilia in the blood of guinea pigs,8, ” it is not able to reproduce the degree of extravascular eosinophilia which follows an immune reaction. It is possible that histamine may under some circumstances (e.g., large doses) activate the same intermediate which immune complexes do, but it appears unlikely that it itself is the mediator which causes substantial extravascular eosinophilia. The results obtained with various routes of antigenic challenge indicate the importance to be attributed to the site at which the antigenic stimulus is applied in determining the locus at which eosinphils accumulate in sensitized animals. The route of sensitization may be intraperitonealg or subcutaneous. In both instances, antibody localizes in the abdominal wall and complexes with the antigen, when reintroduced; only the ant,igen which escapes from the peritoneal cavity is free to react at other sites. If the reaction occurs in the blood, the resultant complexes are clcarcd rapidly by the reticuloendothclial system.32 The site at which the antigen is introduced determines the site at which eosinophils accumulate because this is where the first and probably largest amount of immune complexes are generated and where they persist, longest. Thus, significant peritoneal cosinophilia occurred only when the antigen was introduced at this site. Even when billions of leukocytes were pouring into the peritoneal cavity as the result of nonspecific stimulation with casein, the eosinophil response was insignificant. In t,he one instance in which a relatively large number of eosinophils entered the peritoneal cavity, the cosinophils represented only 1 per cellt of the population, the usual proportion found in the blood of normal animals. It seems evident that the reaction which elicits eosinophilia occurs predominantly at the site of entry of antigen, and that the eosinophilia is the result of a specific immune reaction and not the consequence of a nonspecific inflammatory reaction. SUMMARY
1. Aqueous extracts of the peritoneal lining of guinea pigs which have been sensitized to a foreign protein contain a substance which, when mixed with the sensitizing antigen, evokes peritoneal eosinophilia in recipient guinea pigs. This substance has the properties of antibody, since the extracts contain demonstrable antibody, and since the activity of the extracts disappeared when the antibody was absorbed by the specific antigen.
,542
Norember--December,
J. Allerav 1962
2. Eosinophilia did not occur in the peritoneal cavity, in the skin of the back or in footpads when 0.1 to 100 ,LL~of histamine was injected at these sites. An eoxinophil response was occasionally seen in the blood stream following the intraperitoneal injection of large doses ot’ histarnincl, namely, 10 ,LL~and greater. 3. In guinea pigs sensitized to protein antigens by the intraperitoneal rout,e, peritoneal cosinophilia resulted when an antigenic challenge was administered intraperitoncally, but not when it, was administered intravenously. The injection of casein into the peritoneal cavity of the same animals elicited an influx of several hundred million lcukocytcs, hut the exudate contained only an insignificant number of cosinophils. The Sam? results were obtained when intravenous injection of antigen accompanied the intrapcritoneal injection of casein. 4, The results are interpreted as further evidence that immune complexes mediate the occurrcncc of extravascular cosinophilia. The sites at which eosinophils accumulate are the sites at, which immune complexes are formed iUld persist. Histamine is probably not a major mediator of eosinophilia, at least in guinea pigs. The gratefully
excellent technical acknolr-lcdged.
assistance
of
Miss
Daisy
Killer
and
Miss
Elizabeth
Tappolet
is
REFERENCES 1. Schwarz, E.: Die Lehre von der allgemeinen und iirtlichcn “Eosinophilie,” Ergebn. d. allg. u. path. Anat. 17: 137, 1914. 2. Ringoen, A. R.: Eosinophil Leukocytes and Eosinophilia in Handbook of Hematolog,v, edited by H. Downey, New York, 1938, Paul B. Hoeber, Inc., p. 179. 3. I)zury, D. S., and Cohen, S. G.: Experimental Eosinophilia. Studies on the Origin and Relationship of Tissue Eosinophil Cells to Peripheral Blood Eosinophilia, J. ALLERGY 29: 340, 1958. 4. SDeirs. R. S.: A Theory of Antibodv Formation Involving Eosinophils and ReticuloA endothelial Cells, gature 181: 68i, 1958. I5 Samter, M., Kofoed, M. A., and Pieper, W.: A Factor in I,ungs of Anaphylaetically Shocked Guinea Pigs Which Can Induce Eosinophilia in Normal Animals, Blood 8: 1078, 1953. L.: Experimental Eosinophilia. III. Regional G. Cohen, 8. G., Kantor, M., and Gatto, T,ymph Node Responses to Reactions of Tissue Sensitization, J. ALLERGY 32: 214, 1961. Studies in Experimental Eosinophilia. III. The Induction of Peritoneal Eosino7. Litt, M.: philia by the Passive Transfer of Serum Antibody, J. Immunol. 87: 522, 1961. 8. Vaughn, J.: The Stimulation of the Eosinophil Leueocyte, .T. Path. Bact. 64: 91, 1952. 9. T,itt, M.: Studies in Experimental Eosinophilia. I. Repeated Quantitation of Peritoneal Eosinophilia in Guinea Pigs by a Method of Peritoneal Lavage, Blood 16: 1318, 1960. 10. Speirs, R. R.: The Principles of Eosinophil Diluents, Blood 7: 550, 1952. 11. Hollande! A-Ch.: Enrichissement du liquide fixateur de bouin en acide picrique, par addition d’acktate neutre de cuivre, Compt. rend. Sot. Biol. 81: 17, 1918. 12. Dominici, RI.: Sur une methode de technique histologique apropriee B I’&ude du systhme h&natopo%tique, Compt. rend. Sot. Biol. 64: 221, 1902. 13. Litt, RI.: Studies in Experimental Eosinophilia. II. Induction of Peritoneal Eosinophilia by the Transfer of Tissues and Tissue Extracts, Blood 16: 1330, 1960. Effect of Horse Serum, Adrenal Hormones! 14. Panzenhagen, B. A., and Speirs, R. R.: and Histamine on the Number of Eosinophils in the Blood and Peritoneal Fluid of Mice, Blood 8: 536, 1953. Understanding of the Function of 15. Speirs, R. S.: Physiological Approaches to an E:osinophils and Basophils, Ann. New York Acad. SC. 59: TOG, 1955. 16. Feltlberg! IV., and Miles, A. A.: Regional Variations of Increased Permeability of Skin (:aplllaries Induced by a Histamine Liberator and Their Relation to the Histamine Content of the Skin, J. Physiol. 120: 205, 1953.
Volume 33 Number 6 17.
STUDIES
Perry,
19. 20. 21. 22. 23. 24. 2.5. “G. 27. 38. 29. 30. 3 1. 3 2. 33. 3-f.
EXPERIMENTAL
EOSINOPHILIA.
IV
543
W. L. RI.: Ciba Symposium on Histamine, edited by G. E. W. Wolstenholme and M. O’Connor, Boston, 1956, Little, Brown & Co., p. 342. Archer, R. K.: The Eosinophilic Response in the Horse to Intramedullar; and Intradermal Injections of Histamine, ACTH and Cortisone, ,T. Path. Ract. 72: 8i, 1956. Archer, R. K.: The Mechanism of Eosinopenia by AC’TH and (‘ortiroids in the Horse, .T. Path. Bact. 74: 381. 1957. Archer, R. K.: Eosinophil ‘Leucocytes and Their Reactions to Histamine and 5Hytlroxytryptamine, .T. Path. Bact. 78: 95, 1959. Archer, R, K.: Eosinophil T,eucocyte-attracting Effect of Histamine in Skin. Nature 1ti7: 155 l!l~iO. Archer, R. I<.: Stutlies With Eosinophil Leucocytes Isolated From the Blood of the Horse, Brit. .J. Haematol. 6: 219, 1960. Weir, W. H.: Eosinophilia in Pelvic Lesions and in the Vermiform Appendix, Am. .J. bed. SC. 125: i4; 1903. Opie, E. L.: An Experimental Study of the Relation of Cells Tvith Eosinophile Grauulation to Infection With an Animal Parasite (Trichina snirctlis). I I Am. J. Med. He 127’ 177 1’W. Vaughd, Jl:’ The’ Filnction of the Eosinophile Leukocyte, Blood 8: 1, 1953. Rchild, H. 0.: Ciba Symposium on Histamine, edited by G. $1. \i:\‘. Wolstenholme aucl C. M. O’Counnr. Boston. 1956. Little. Brol~-n & Co.. D. 139. Campbell, D. H.: delationship ot! the l?osinophil Reip;nse to Factors Involved in Anaphylaxis, J. Infect. Dis. 72: 42, 1943. Ramter, M.: The Response of Eosinophils in the Guinea Pig to Sensitization, Anaphl-laxis and Various Drugs, Blood 4: 217, 1949. Knott, F. A., and Pearson, R. 8. IX: Eosinophilia in Allergic Conditions, Guy’s Hosp. Rep. 84: 230, 19%. Kline, B. S., Cohen, X. B., and Rudolph, J. A.: Histologic (‘hanges in Allergic anti Nonallergic Wheals, .I. ALLERGY 3: 531, 193”. Weinberg, M.: anti SPguin. P.: Recherches biologiques sur l’tiosinophilie, arm. Inst. Pasteur 28: 4i0, 1914. Biggart, J. H.: Some Observations on the Eosinophile Cell, .J. Path. Bact. 35: 799, 19X. Haj6s, K.: BeitrLge zur Eosinophiliefrage. II. Die Vertrilung der eosinophilen Zcllen nach Protrininjrktionen und im anaphylaktisierten Mwrsrhwcinclwn, Ztschr. gcs. exuer. Med. 59: :183. 1X.X \Veigle; W. 0.: Elinrina&on of Antigen-Antibody Complexes From Sera of Rabbits, J. Immunol. 81: 204, 1958. C.
18.
IN
IN EXPERIMENTAL
IV. Determinants Mortimer
Litt,
of Eosinophil M.D.,*
Boston,
EOSINOPHILIA
Localization Mass.
well established that tissue eosinophilia is not necessarily associated with blood eosinophilia.l, 2 The reasons for this dissociation are not clear, part,icularly if one considers that the bone marrow is the major, though not the only, site of cosinophil formation, and the blood stream is the vehicle by which these cells are transported to the tissues.2, 3 Most investigators agree that eosinophilotactic stimuli commonly arise at tissue sites (rather than in the blood stream), but the nature of the stimulus has been variously depicted. It has been held that this stimulus is (3.) an antigeq4 (2) a product of antigen-antibody union,“, (i (3) the antigen-antibody complex itself,7 and (4) histamine (one of the products of immune reactions) .’ This paper is concerned with the fact,ors which result in the localization of cosinophils at cxtravascular sites, and addresses itself to three subjects : (1) the identification of a transferable tissue factor which elicits eosinophilia, (2) the possibility that histamine is one of the mediators of the eosinophil response, and (3) the relationship between the site of challenge and the site of eosinophil infiltration.
I
T IS
MATERIALS
AND
METHODS
Guinea Pigs.-Female Hartley-strain guinea pigs, weighing 275 to 300 grams, were obtained from local breeders and fed Rockland rabbit pellets ad libit,um, supplemented by cabbage three times a week. Eosinophil Counts.-Peritoneal: The method of inducing peritoneal eosinophilia by repeated injections of foreign protein solutions, and the method of quantitating the eosinophil response by peritoneal lavage, have been described previously.” Blond: The ear veins were dilated by warm air from a hair dryer and nirk(d with a scalpel blade. Free-flowin, w blood was diluted 1:20 in a, 1 per cent l?rorn tbe Charming Laboratory, Boston City Hospital, and the Department of Bactcriand Immunology, Harvard Medical School, Boston, Mass. This work was supported by Grant DRG-588 from the Damon Runyon Memorial Fund for Cancer Research and Grant No. 506 from the Greater Boston Chanter of the Massachusetts Heart Association. Received for publication Sept. 26, 1962. *Research Fellow of the Helen Hay Whitney Foundation.
oloay
532
corrected white blood cell pipette with an acetone diluent,lO shaken for 30 seconds, and counted in an hcmocytomctcr. Counts were recorded to the nearest 10 cells.
Total Leukocyte Cozcnts.-The diluent was 2 per cent acetic acid. With the cascin-induced exudates, clotting was occasionally a problem. This was handled 1))~simply diluting the exudate at least 1 :I0 with saline before taking a sample for further dilution in the leukocyte-diluting pipette. Tissue Eosinophilia.-Skin: The hair over the dorsal aspect of the animals was rcmorcd by coarse and fine electric clippers, followed by an electric shaver. Six sites, one square centimeter in size and at least 2 cm. apart, were marked in ink, and intracutaneous injections through 27 gauge needles, were made at four of the sites. All six sites were subsequently removed, fixed in Bouin-Hollande,” embedded in paraffin, sectioned at +.L, and stained with Dominici.l* The total slumber of eosinophils in cross sections of the skin site (two sections, 4 mm. apart, were examined for each site) was determined by direct microscopic count. Footpads: Injections were made into both right footpads; the left footpads served as controls. The footpads were subscqnentlp removed and processed in the same way as the skin. Chenzicals.-Histamine diphosphate* was dissolved in 0.85 per cent saline. All amounts are expressed as free base. (In order to facilitate comparisons when citations are made of the literature, all doses of histamine have been con\-erted, when necessary, to the amount of histamine base; the conversion factors were 0.6 for histamine dihydrochloridc and 0.36 for histamine phosphate.) Horse serum was obtained from the Commonwealth of Massachusetts Department of Public IIealth Antitoxin and Vaccine Ilaboratory, Boston, Massachusetts. Hemocyanin was prepared as described previously.” Tprodc’s solution had a modified formula, described previously.13 Prepnmtion of Guinea 1%/s for Peritoneal Studies.-The method employed has been described in detail previously. I3 Essentially, each guinea pig was given weekly intraperitoncal injections of either 1 ml. horse serum or 0.5 ml. of 0.5 per cent hemocyanin for about 4 months, at which time challenge with antigen regularly produced peritoneal eosinophilia. When the eosinophil response to the last preparatory injection of antigen had ceased, the ant,igen was administered 1,~ a new route: intravenously in a le,e vein or subcutaneously in the ventral abdomen, either alone or accompanied by a simultaneous intraperitoneal injection of 8 ml. of sterile I:! per cent sodium cascinatc:t Some animals were tested with intrapcritoneal cascin alone. Whcncver a particular animal scrvcd as subject for testing more than one type of material, an int,raperitoncal challenge with antigen was interposed to verify that the animal was still in a state of responsiveness to the usual cffectirc stimulus (intraperitoneal antigen). *Pfanstiehl f~utritional
Laboratories. Biochcmirals
Inc., XVaukegan, Corp., Cleveland,
Illinois. Ohio.
534
November-December,
J. Allergy 1962
Those guinea pigs which were inoculated exclusively by the intracutaneous I-onte wwived 0.1 ml. of 0.25 per ccrrt h(~rrrocy:rrrin in each ventral abdominal quadrant each week. E’r~pnmtion cud Alssc~y of fCc:xtmcts of Peritoneal Lining.-Ten sensitized guinea pigs serrcd as tissue donors. Sftrr it, had been ascertained that, intraperit,oneal hemocyanin evoked a peritoneal eosinophil response, the guinea pigs wwc sacrificed (7 to 1-l days after the last injection). The peritoneal lining was removed, rinsed in two 10 ml. changes of Tyrodc’s solution, cut into 1 cm. strips and shaken in Tyrode’s solution for 4 hour~s at 37’ C. The strips were removed by filtration through cotton gauze and the liquid portion was centrifuged at 10,000 r.p.m. in a X3-1 Scrvall ecntrit’ugo. One hundred and fift,y milliliters of clear supernatant was frozc:n until USA. The extract, was tested in vitro for antibotl~ and in viva for eosinophilotactic activit,y both before and after absorption of the antibody by hernocyanin. The in yi1.o tests wer~c conducted 1)~ mixin, cr 7.5 ml. of extract with 0.5 ml. of 0.5 per cent hemocyanin, incubating for 30 min. at 37O C., and injecting the mixture into the peritoneal cavity of animals preparcd with horse serum (which is non-cross-reacting in this system) : total peritorrc~al eosinophil counts wcrc ma&~ 2-l hours following injcctiori.” The tests for antibody consisted of interfacial precipitation with antigctn in capillary tubes and double diffusion in agal’ gel against antigen; in the Iattc>r cast’, two distinct. bands formed after 21 hours. Srvcnty-t\vo millilitc~rs of the ext,ract was absorbed by two sequential additions of 1 ml. of 11 per cent heniocyanin. :jftcr ~a(~11addition, the solution was incubatetl for :! hours at 3’ (1.> and I)Iacctl at 3’ (‘. for 7 days. The resultant precipitate was rcmoyc>cl by cent rifugation. I~‘ollowing the sc~ontl portion of antigen, tests of suI)crnatarrt showed antigen in CSWSS.Both antigen and any soIublo complexes whic~lr nright Iiavc becir pr(lsclnt \v~‘r(l ~rtro~c~l by ccnt,riThe fugation at 37,000 r.p.m. Jc)r 2 hours irr a Sl)inco JIotl~l T1ult~accntrifugc. wp~~rnstnnt contain& neither amtig(ln nor+ arrtibotlp. as tlctcrwinetl by ring test ant1 diffusion in agar gel (2 weeks).
ophiZia.-It has been rcportcd”; that an extract of sensitized peritoneal lining, in association with the sensitizing ant,igrn, could induce peritoneal eosinophilia ; the active material in the extract was not identified. Table I shows that such an extract, which was active (when associated with antigen) in producing peritoneal eosinophilia, lost this activity when its antibody content was removed by specific absorption with antigen. The unabsorbed extract produced eosinophilia in 7 of 10 recipients, whereas the absorbed extract was inactive in all of 7 recipients. ‘1. The
Possible
Role
of
Nistcrmi~~~
as
(I XecLiintor
of
the
Eosinophil
Ir’es2)onsc,.-~-Tlrtt striking parallelism lwtwwn the c~ircumstances which result in lristarninc~ release and those which result in cosinophilia provide ample reason to consider histamine as a possible mediator of eosinophilia. If histamine does
Volume 33 Number 6
STUDIES
IN
EXPERTMENTAL
EOSINOPHILIA.
1V
535
TABLE J. EOSINOPJIILOTACTIC ACTIVITY OF EXTRACTS OF PERITONEAL LINING FROM GUINEA PIGS SENSITIZED WITH HEMOCYANIN GUINEA PIG I 11
Response
to
sensitizing
12 I3 Unabsorbed 8” 3
2 to
6
protein 22
Response
I4 Extract
extract
21
26
23
15
I6
I7
I8
6 4 3 6 1 ml. horse serum 39 12 17 25
I9110 8
6
6
22
40
21
4
0
5
2
3 4 4 1 0 1 5 7.5 ml. extract + 0.5 ml. - 0.5% hemocyanin 10 10 10 6 18 14 12
GUINEA PIG 11 1 12 1 13 j 14 I 15 1 16 1 17 1 18 2 Response
to
sensitizing
protein 18 5
Response
to
Absorbed 3
extract 7
13
Extract 6 3 5 6 3 1 ml. horse serum 18 18 14 43 20
5 2 4 2 4 7.5 ml. extract + 0.5 ml. - 0.5% 4 3 3 2 6
1 13
6 3 hemocyanin 2 1
*Each set of ~‘esponses shows the total number of eosinophils in millions, which wel‘e present in the peritoneal cavity immediaiely before and 24 hours following each injection. The recipients had been prepared by 4 months of weekly intraperitoneal injections of 1.0 ml. horse serum.
mediate eosinophilia it should be possible to bypass the long induction period which is typical of the usual method for inducing experimental eosinophilia. Instead of requiring weeks of repeated injections of foreign protein, eosinophilia should occur soon after the administration of histamine. A series of experiments was carried out in which the consequences of histamine administration were examined at each of four sites in guinea pigs. In the peritoneal cavity: Histamine was injected into the peritoneal cavity of guinea pigs and the total peritoneal eosinophil count was determined 24 hours later. This interval was chosen because, when sensitized guinea pigs are challenged by antigen, the peak accumulation of eosinophils occurs in about 24 hours.” The doses of histamine were 0.1, 1.0, 10, and 100 pg ; 4 guinea pigs were injected with each dose. No cosinophilia resulted in any case. The mean number of eosinophils found was 1.5 million and the range was 0 to 3 million. This is comparable to t,he number, 2.7 & 1.0 million, found in normal uninjected guinea pigs. o Since the duration of action of parenterally administered histamine is relatively short, it was possible that by 24 hours, when the assay was performed, the response was already over. In addition, the response might require contact with histamine that is more prolonged than that provided by a single dose. Accordingly, another group of 16 animals was given the same doses of histamine, but in a course of injections; the dose was repeated every 8 hours for a total of three doses, and the assay was made 8 hours after the last injection. Again, no eosinophilia resulted. This time the average cosinophil count was 0.25 million, and the range was 0 to 1 million. In skin: In order to determine whether hist,amine could attract eosinophils into skin, histamine was injected in the posterolateral wall of a group of 30
T11’1’ ‘1’
536 TABLE
II.
GUINEA
BLOOD
PIG
EOSIKOPHILIA*
1
IN
Qurmn
PIGS Fo~r,owxm HISTAMINE IiOlJItS
i-0
J& 0
10 10
November--l)ecemher,
TNTIMPER~TONEAL ALTER
10 0 10 10 0 10 10 10 10 30 0
10 40 90 10 30 10 80 IO 90 0 0
:: 20 21
10 10 10 30 0
50 40 40 50 10 10
80 50 40 :iO 50 10 0 30 0 0
100 50 0 30 10 0 0 30 0 0
22 23 24 25 26 27 28 29 30 31
30 CO 90 30 40 60 0 IO 90 50
ii 60 10 0 50 0 10 30 10
50 130 40 30 40 30 0 40 130 40
50 90 40 180 100 50 0 0
30 IO 40 50 30 0 30 50 40 40
40 40 50 50 20 50 40 100 20 20
150 30 40 50 10 60 40
150 10
2
0
4 5 G
0 0 0 0
180 100 50 30 0 50 30 30 I0
7 8 9 IO
11 12
10 50 30 80 0
13 14 15
1 1 1
1s 17
5 5 5
5 5 5 5 5 5 5 10
32 33 34 35 36 37 38 39 40 41
10 10 10 10 10 10 10 10 10 50 50 50
42 43 44 45 46 47 48 411 50 51
50 30 50 50 50 50 50
100 100 100 100
52 53 54 55 56 57 58 *lLkminophils
lili 40 60
100 100 100 per
cubic
10 60 10 30 0 JO 190 40 0 90 1;:
100 millimeter.
30
180 10 60
190 10 10
10 310 40 0
IO 280
10 30 150 100 0
100
80 10
::
ii 60 0 50 150 190 30 0 450 430 40
50
40 50 130 2x0 X0
2 150 300 450 '30
250 80 50 (I 60 280
MO 100 50 CI 490 250
190 130 100 30 930 50 640
IXJECTIOS
OF
IN,JIXTION
I_ 30 0 90 30 30 10 10 10 10 30 0
J. Allerpr 1962
30 100 80 30 fin 160 30 90 80 0 0 140 10 100 130 3-n 40 10 40 6: zi 10 40 50 30 0 60 90 50
Z-1 10 30 30 80
/
4x 10 90 ii
10 10
0 30
1.4: 1 A0 0 0
IO 60 180 40 0
10 30 30 50 50 50
50 40 0 360 40 50
3: 50 0
10 90 30 RO
140 40 140 40 ii
100 60 40 80 30 180 10
1: 10 60
1:: 130
480 190 60
0 30 100 50 50 60 110 50 30
30 50 40 80 0 50 40 40 10
100 430 340 30 160 140 210 350 530 240
10 130 80 10 30 100 40 100 160 80
130 10 50 80 10 140 80 50 50 10
130 60 130 80 1,010 530 490
260 '0 ;'O 30 350 200 450
50 80 40 10 60 40 40
630 50 0 50
10 250 130
Volume 33 Number 6
STUDlES
IN
EXPERrMENTXL
TABLE
GIJINEA
PIG
HISTAMINE (P!T)
EOSINOPHILIA.
II.
Cont’d
2
/
HOURS 0
59
100
60
:: 6% 63 64
100 100 100 100
.50 10 60 50 0
)
340 60 30 80 60 0
4
AFTER j
6
130
700
30 10 110 40 50
100 "00 30 40
537
IV
INJECTION 112
/
1,340 30 50 50 140 190
24 560 600 50 40 40
/
4X IO 10 30 10 110 0
guinea pigs. Each animal received four different doses-O.& 1.0, 10, and 100 pg-each at a different skin site, and groups of 5 of these similarly treated animals were sacrificed at 1, 2, 4, 8, 12, and 24 hours, respectively. The skin sit,es were removed for histologic cxsminabion. Eosinophils were found in only three of the 180 sites (120 experimental, 60 control) and numbered only 1, 4, and 2 per section (the latter was an uninjected site). In footpads: Similar experiments were carried out, in which the injections were made into footpads. Groups of 5 guinea pigs each were injected in both right footpads and all 4 footpads were subsequently removed. The first group of animals received one microgram of histamine at each injection site, the second group received 10 pg, and the third, 100 pg. The sites were examined 24 hours following injection and no eosinophils were found in any of the sections. The experiment was repeated, except that, the specimens were removed 4 hours after the injections. In nine of the thirty injected sites, a few neutrophils were found along with between one and eight eosinophils. In the hlootl stwcrnt: In studying the effects on the blood eosinophil count of an intraperitoneal injection of histamine, the experiments of Vaughn8 were repeated, except that t,he dose of histamine covered a loo-fold range. A group of 11 noninjected guinea pigs served as controls and they demonstrate (Table II) the degree of fluctuation which may be encountered in the course of collecting blood samples over a 48 hour period: the average starting count was 47 per cubic millimeter, while individual counts during the ensuing 2 days ranged as high as 180 per cubic millimeter.* Another group, consisting of 53 guinea pigs, with an average zero count of 41 per cubic millimeter, was injected with various doses of histamine, ranging from 1 to 100 pg. There was no apparent response to either the 1 pg or 5 pg dose, except for a single isolated high value (360 per cubic millimeter) at 48 hours in the case of the 1 ,LL~dose. With higher doses, there were definite responses. Four of the 10 guinea pigs which received the 10 pg dose had, at one time or another, an clcvated count, ranging from 190 to 630 per cubic millimet,er. A fivefold greater dose, 50 pg, resulted in elevated counts in 6 of the 10 recipients; thcsc counts ranged from 230 to 430 per cubic millimeter. The highest, dose, 100 pg, resulted in responses in 7 *The group means at any particular time and dose exhibit very large standard deviations. This reflects the fact that there at-e two classes of guinea pigs: responders and nonresponders. If the results are pooled one would conclude that there are no statistically valid effects. Yet it is grossly evident that some injected animals attain abnormally high values. Therefore. the results are prfxente~l as obtained in individual animals. A count greater than 180/mm.3 was considered abnormal. It is the highest value obtained in the control group and is about thrw standard deviations above the nlean of all the control counts. The normal values cited in the literature COVVI a wide range and the importance of working with young animals and using internal standards has been emphasized.5
53x
November-December.
J. Allergy 1962
of 70 recipient,s, and the level of response was higher; 4 animals had at least one count in excess of 500 per cubic millimeter, two of which exceeded 1,000 per cubic millimeter. In general, the initial response was noted as early as 2 hours, with the peak responses coming at 6 or 12 hours. All reaction had ceased (with the one possible exception already noted) by 48 hours. 3. The Relationship Between the Bite of Challenge and the Site of Eosinophi1 Infiltration.-Eosinophilia has been induced experimentally in a wide variety of tissues : conjunctivae, peritoneal cavit,y, skin, blood stream, muscle, lung, intestine. 2, 9, 15, 24, 31, 32, 33 While the sensitizing injections may take place at various loci, the predominant eosinophilia takes place at the site of deposition of the challenging dose of antigen. It is not clear whether the specific cosinophilotactic stimuli are generated at the challenge site or whether nonspecific inflammatory stimuli result in local cell accumulations during a generalized systemic reaction. The following experiments were undertaken to explore this question. In the peritoneal cavity, eosinophilia will result from repeated injection of foreign protein.9 Following a course of weekly intracutaneous injections into the ventral abdominal wall, the same result ensued. By the fourth injection of hemocyanin (0.5 ml. of 0.5 per cent solution), 9 of 16 guinea pigs had developed peritoneal eosinophilia; the average value was 19 million and the range was 13 to 27 million. In guinea pigs which have been sensitized by the intraperitoneal route, a challenging inject,ion of antigen results in peritoneal cosinophilia when it is administered intraperitoneally, but not when it is given intravenously (Table III). Nonspecific inflammatory factors, associated with the intraperitoneal injection might have accounted for the localization of eosinophils. Such animals have increased stores of eosinophils in their bone marrow,9 which might be “swept out” as part of a general leukocytic response. However, this is not the case, since the introduction of casein into the peritoneal cavity, while it evoked a striking leukocytic response in the peritoneal cavity (944 million to 2.4 billion total leukocytes), did not evoke an eosinophil response: less than 1 per cent of the leukocytes were eosinophils and their total number (0 to 9 million) was very small compared to t,he response which occurs upon injection of the specific sensitizing antigen. However, in the absence of antigen, few eosinophils might be released, making few available to be localized nonspecifically. Therefore, the sensitizing antigen was reintroduced, but by the intravenous route; thus it could act in the blood stream, or at any site served by the blood stream. Simultaneously, rasein was introduced into the peritoneal cavity, in order to encourage the localization of large numbers of leukocytes. The resultant peritoneal exudate contained large numbers of leukocytes ranging from 30 million to 6.8 billion. Yet, in spite of these apparently farorablc circumstances, few of the cells in the exudate were eosinophils. The total number of eosinophils did not approach what was found in the same animals upon peritoneal cha.llenge, and, in fact, the numbers were virtually what one finds in a normal, unstimulated animal.” Thcrc were two exceptions : guinea pig No. 9 had about as many cosinophils in tht> esudate as were found following intraperitoneal ant,igenic stimulation, but only
STUD1ES
L’olume ii Number 6
1N EXPERIMENTAL
EOSINOPHILIA.
IV
539
0.2 per cent of the total cells were eosinophils, whereas following intxapcritoneal antigenie stimulation, 25 per cent were eosinophils. In the case of guinea pig No. 8, the only one which showed a substantial nnmber of eosinophils (57 million), only 1 per cent of the total cells were eosinophils, as against about a 45 per cent eosinophilia followin g intrapcritoneal challmge with antigen (in the absence of casein). In a single instance, the antigen was administered subcutaneously, along with a simultaneous intraperitoneal iqjection of casein. Here too, there was no significant eosinophil response. TABLE
TII.
E:FFECT
OF THE ROUTE PERITONEAL
OF ANTIGENIC ~OSINOPFIlI~IA
HTIM~~ULATIO~~~ ON THE IN GUINEA PIGS GIJIiYEh
1.1’. ROI’TE
(‘ASEIN
OCWRREK~E
OF
PIG
1~2/3/4~516,7~819
IT.”
30t
17
20
15
16
45
40
30
10
I.V."
76 3
120 4
50 1
45
50
105
90 1
75
40
--_ 15
26
210 4
9
0
t I .v. is. c . *
0
t t
8 114 -
12
30 -
-
340
9 _920
-
-
4 1,650
944 2,400 2,200 4 8 57 12 ___-~ 200 6,800 6,000 6,000 6 900
Guinea pigs l-6 were prepared hemocyanin. The preparatory and Guinea pigs 7-S were prepared horse serum. The preparatory and Determinations were made 24 *I.P. = intraperitoneal. I.V. = tNumerator denotes millions Denominator denotes millions
by 4 months of weekly intraperitoneal challenging dose was 0.5 ml. of 0.5’jb solution. by 4 months of weekly intraperitoneal challenging dose was 1 ml. of horse serum. hours after the challenging injection. intravenous. S.C. = subcutaneous. of peritoneal eosinophils . of peritoneal leukocytes
injections
of
injections
of
DISCUSSION
Active sensitization of guinea pigs by the intraperitoneal route resulted in an accumulation of eosinophils in the peritoneal cavity and the localization of antibody in the lining of the cavity. This antibody, when extracted, complcxcd with antigen, and passively transferred int,o the peritoneal cavity of other guinea pigs, resulted in the rapid mobilization of eosinophils in the peritoneal cavity of the recipients. The activity of the extracts reported here and previouslyl” is related to the antibody content of the extracts, since removal of the antibody by precipitation with specific antigen abolished the activity. A similar activity in the serum of sensitized animals had previously been attributed likewise to its antibody content.’ While this earlier finding, that immune complexes containing serum antibody induced eosinophilia, suggested t,hat such complexes might mcdiatc the emigration of cosinophils from their bone marrow stores into the blood stream, it could not account for the more prominent localization at the site of introduction of the antigen. The current findings indicate that the mechanism of experimental tissue eosinophilia, as commonly
540
LITT
November-December,
J. Allerry 1962
produced by repeated injections of foreign protein, includes two stages: the preparatory injections of antigen result in the production of antibody and its localization at tissue sites, while the challenging injection of antigen combines with tissue-stored antibody. The resultant immune complex attracts the eosinophils. It seems possible that the activity which Samter, Kofoed, and Pieper” found in the lungs of guinea pigs who were anaphylactically shocked was also related to an immune complex. While Cohen, Kantor, and Gatto” found that the introduction of antigen and antibody at separate sites in the footpads of rabbits resulted in eosinophilia in the draining lymph node, they found t,hat irnmune complexes formed in vitro wcrc inactive in this respect. On the basis of these observations, they concluded that eosinophilia is not mediated by t,he immune complex itself, but rather by sonic product of the interaction of antigen, antibody, and some tissue component. Because of the differences in species, antigen, and route, the above experiments arc not strictly comparable with ours. Histamine is probablv not a major mc~tliator of the eosinophil response. It did not provoke an accumulation of cosinophils at any of the cxtravascular sites at which it was introduced. The results of the peritoneal studies are in accord with those of I’anzcnhagen and Spcirs,17I4 ~vho reported that 18 pg of histamine caused no eosinopbilia in the peritoneal (aa\-it,>-of mice, while 72 pg caused only a slight increasc.l” Relatively little histamine is stored in the skin of guinea pigs* and only insignificant amounts of histamine are released during allergic skin reactions,l’ yet even if all of the histamine were activated, it could not account for eosinophilia-one hundred t,imes t,he total cndogenous amount failed to produce any eosinophil responscl. In the horse, on the other hand, the intracutaneous injection of hist,amine does result in a local accumulation of eosinophils, but the response is substantial only with large doses of histamine. Thus, 900 ,~g,l~ 90 pg,l’ and 720 ,~g~” of histamine create foci in which the average coneentrat)ion of eosinphils is about 20 per high-power microscopic field. Ilower doses result in a mild eosinophilia: with 36 ,.~g$,the concentration averaged 6 to 7 per high power field,“l and with 3.6 pg, the concentration averaged 4 to 5 per high-power field.“’ AS Archer points out, presumably no local concentration of histamine approaching the level of 72 pg could occur in life, and it is questionable whether lower amounts occur. Yet even ~creral hundred micrograms of histamine does not reproduce the degree of eosinophilia which occurs in allergic humans and in experimental animals.““, 24 The results of the blood studies confirm those of Vaughn83 25; an eosinophil response occurred in the blood stream of some guinea pigs given large doses of histamine, but these were amounts of histamine which are not likely to bc encountered ill vivo. The cffccti\c doses, 10 pg and gr’cater, are larger than the amounts which arc activated by an alltbrgic event. Hven lethal anapbylaxis releases only a few micrograms of histamine: about ‘1 pg arc rclr:~ed from the *A footpa< contains about ~mrl), and 1 cmp of dorsal skin X 240 mg. per site).
1.2 pi’ of histamine (an injection site)
(i.7 gg contains
per gram’6 X 3 60 mg. per footabout 0.7 pg (2.9 86 per gra~rlx
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lung.2G Contrary results have been reported when somewhat different experimental designs were used. Campbcll27 found no rise in the cosinphil count after hc administered 500 ,~g of histamine intral)c~ritoncallv~all~ thrice daily for 3 days and sampled the blood every 12 hours. SanltW” also found that histamine (75 pg intracardially) did not induce eosinophilia. in normal animals 20 hours after injection; however, in animals sensitized to horse serum t,hc injection of histaThis parallels the situation in human mine alone did result in eosinophilia. beings in whom histamine alone provokes little eosinophilia unless the subject is allergic, in which case a significant cosinophilia occurs in the wheal produced by an intracutaneous injection of histamine.‘g, 3n Thus, while histamine can cause minor accumulations of eosinophils in the skin of horses18-22 and substantial eosinophilia in the blood of guinea pigs,8, ” it is not able to reproduce the degree of extravascular eosinophilia which follows an immune reaction. It is possible that histamine may under some circumstances (e.g., large doses) activate the same intermediate which immune complexes do, but it appears unlikely that it itself is the mediator which causes substantial extravascular eosinophilia. The results obtained with various routes of antigenic challenge indicate the importance to be attributed to the site at which the antigenic stimulus is applied in determining the locus at which eosinphils accumulate in sensitized animals. The route of sensitization may be intraperitonealg or subcutaneous. In both instances, antibody localizes in the abdominal wall and complexes with the antigen, when reintroduced; only the ant,igen which escapes from the peritoneal cavity is free to react at other sites. If the reaction occurs in the blood, the resultant complexes are clcarcd rapidly by the reticuloendothclial system.32 The site at which the antigen is introduced determines the site at which eosinophils accumulate because this is where the first and probably largest amount of immune complexes are generated and where they persist, longest. Thus, significant peritoneal cosinophilia occurred only when the antigen was introduced at this site. Even when billions of leukocytes were pouring into the peritoneal cavity as the result of nonspecific stimulation with casein, the eosinophil response was insignificant. In t,he one instance in which a relatively large number of eosinophils entered the peritoneal cavity, the cosinophils represented only 1 per cellt of the population, the usual proportion found in the blood of normal animals. It seems evident that the reaction which elicits eosinophilia occurs predominantly at the site of entry of antigen, and that the eosinophilia is the result of a specific immune reaction and not the consequence of a nonspecific inflammatory reaction. SUMMARY
1. Aqueous extracts of the peritoneal lining of guinea pigs which have been sensitized to a foreign protein contain a substance which, when mixed with the sensitizing antigen, evokes peritoneal eosinophilia in recipient guinea pigs. This substance has the properties of antibody, since the extracts contain demonstrable antibody, and since the activity of the extracts disappeared when the antibody was absorbed by the specific antigen.
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J. Allerav 1962
2. Eosinophilia did not occur in the peritoneal cavity, in the skin of the back or in footpads when 0.1 to 100 ,LL~of histamine was injected at these sites. An eoxinophil response was occasionally seen in the blood stream following the intraperitoneal injection of large doses ot’ histarnincl, namely, 10 ,LL~and greater. 3. In guinea pigs sensitized to protein antigens by the intraperitoneal rout,e, peritoneal cosinophilia resulted when an antigenic challenge was administered intraperitoncally, but not when it, was administered intravenously. The injection of casein into the peritoneal cavity of the same animals elicited an influx of several hundred million lcukocytcs, hut the exudate contained only an insignificant number of cosinophils. The Sam? results were obtained when intravenous injection of antigen accompanied the intrapcritoneal injection of casein. 4, The results are interpreted as further evidence that immune complexes mediate the occurrcncc of extravascular cosinophilia. The sites at which eosinophils accumulate are the sites at, which immune complexes are formed iUld persist. Histamine is probably not a major mediator of eosinophilia, at least in guinea pigs. The gratefully
excellent technical acknolr-lcdged.
assistance
of
Miss
Daisy
Killer
and
Miss
Elizabeth
Tappolet
is
REFERENCES 1. Schwarz, E.: Die Lehre von der allgemeinen und iirtlichcn “Eosinophilie,” Ergebn. d. allg. u. path. Anat. 17: 137, 1914. 2. Ringoen, A. R.: Eosinophil Leukocytes and Eosinophilia in Handbook of Hematolog,v, edited by H. Downey, New York, 1938, Paul B. Hoeber, Inc., p. 179. 3. I)zury, D. S., and Cohen, S. G.: Experimental Eosinophilia. Studies on the Origin and Relationship of Tissue Eosinophil Cells to Peripheral Blood Eosinophilia, J. ALLERGY 29: 340, 1958. 4. SDeirs. R. S.: A Theory of Antibodv Formation Involving Eosinophils and ReticuloA endothelial Cells, gature 181: 68i, 1958. I5 Samter, M., Kofoed, M. A., and Pieper, W.: A Factor in I,ungs of Anaphylaetically Shocked Guinea Pigs Which Can Induce Eosinophilia in Normal Animals, Blood 8: 1078, 1953. L.: Experimental Eosinophilia. III. Regional G. Cohen, 8. G., Kantor, M., and Gatto, T,ymph Node Responses to Reactions of Tissue Sensitization, J. ALLERGY 32: 214, 1961. Studies in Experimental Eosinophilia. III. The Induction of Peritoneal Eosino7. Litt, M.: philia by the Passive Transfer of Serum Antibody, J. Immunol. 87: 522, 1961. 8. Vaughn, J.: The Stimulation of the Eosinophil Leueocyte, .T. Path. Bact. 64: 91, 1952. 9. T,itt, M.: Studies in Experimental Eosinophilia. I. Repeated Quantitation of Peritoneal Eosinophilia in Guinea Pigs by a Method of Peritoneal Lavage, Blood 16: 1318, 1960. 10. Speirs, R. R.: The Principles of Eosinophil Diluents, Blood 7: 550, 1952. 11. Hollande! A-Ch.: Enrichissement du liquide fixateur de bouin en acide picrique, par addition d’acktate neutre de cuivre, Compt. rend. Sot. Biol. 81: 17, 1918. 12. Dominici, RI.: Sur une methode de technique histologique apropriee B I’&ude du systhme h&natopo%tique, Compt. rend. Sot. Biol. 64: 221, 1902. 13. Litt, RI.: Studies in Experimental Eosinophilia. II. Induction of Peritoneal Eosinophilia by the Transfer of Tissues and Tissue Extracts, Blood 16: 1330, 1960. Effect of Horse Serum, Adrenal Hormones! 14. Panzenhagen, B. A., and Speirs, R. R.: and Histamine on the Number of Eosinophils in the Blood and Peritoneal Fluid of Mice, Blood 8: 536, 1953. Understanding of the Function of 15. Speirs, R. S.: Physiological Approaches to an E:osinophils and Basophils, Ann. New York Acad. SC. 59: TOG, 1955. 16. Feltlberg! IV., and Miles, A. A.: Regional Variations of Increased Permeability of Skin (:aplllaries Induced by a Histamine Liberator and Their Relation to the Histamine Content of the Skin, J. Physiol. 120: 205, 1953.
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W. L. RI.: Ciba Symposium on Histamine, edited by G. E. W. Wolstenholme and M. O’Connor, Boston, 1956, Little, Brown & Co., p. 342. Archer, R. K.: The Eosinophilic Response in the Horse to Intramedullar; and Intradermal Injections of Histamine, ACTH and Cortisone, ,T. Path. Ract. 72: 8i, 1956. Archer, R. K.: The Mechanism of Eosinopenia by AC’TH and (‘ortiroids in the Horse, .T. Path. Bact. 74: 381. 1957. Archer, R. K.: Eosinophil ‘Leucocytes and Their Reactions to Histamine and 5Hytlroxytryptamine, .T. Path. Bact. 78: 95, 1959. Archer, R, K.: Eosinophil T,eucocyte-attracting Effect of Histamine in Skin. Nature 1ti7: 155 l!l~iO. Archer, R. I<.: Stutlies With Eosinophil Leucocytes Isolated From the Blood of the Horse, Brit. .J. Haematol. 6: 219, 1960. Weir, W. H.: Eosinophilia in Pelvic Lesions and in the Vermiform Appendix, Am. .J. bed. SC. 125: i4; 1903. Opie, E. L.: An Experimental Study of the Relation of Cells Tvith Eosinophile Grauulation to Infection With an Animal Parasite (Trichina snirctlis). I I Am. J. Med. He 127’ 177 1’W. Vaughd, Jl:’ The’ Filnction of the Eosinophile Leukocyte, Blood 8: 1, 1953. Rchild, H. 0.: Ciba Symposium on Histamine, edited by G. $1. \i:\‘. Wolstenholme aucl C. M. O’Counnr. Boston. 1956. Little. Brol~-n & Co.. D. 139. Campbell, D. H.: delationship ot! the l?osinophil Reip;nse to Factors Involved in Anaphylaxis, J. Infect. Dis. 72: 42, 1943. Ramter, M.: The Response of Eosinophils in the Guinea Pig to Sensitization, Anaphl-laxis and Various Drugs, Blood 4: 217, 1949. Knott, F. A., and Pearson, R. 8. IX: Eosinophilia in Allergic Conditions, Guy’s Hosp. Rep. 84: 230, 19%. Kline, B. S., Cohen, X. B., and Rudolph, J. A.: Histologic (‘hanges in Allergic anti Nonallergic Wheals, .I. ALLERGY 3: 531, 193”. Weinberg, M.: anti SPguin. P.: Recherches biologiques sur l’tiosinophilie, arm. Inst. Pasteur 28: 4i0, 1914. Biggart, J. H.: Some Observations on the Eosinophile Cell, .J. Path. Bact. 35: 799, 19X. Haj6s, K.: BeitrLge zur Eosinophiliefrage. II. Die Vertrilung der eosinophilen Zcllen nach Protrininjrktionen und im anaphylaktisierten Mwrsrhwcinclwn, Ztschr. gcs. exuer. Med. 59: :183. 1X.X \Veigle; W. 0.: Elinrina&on of Antigen-Antibody Complexes From Sera of Rabbits, J. Immunol. 81: 204, 1958. C.
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