SUPPRESSION
OF T CELL-MEDIATED
BASOPHIL HYPERSENSITIVITY
PIGS IMMUNIZED
WITH
CUTANEOUS
BY S E R U M
MYCOBACTERIAL
FROM
GUINEA
ADJUVANT*
BY E. BRUCE MITCHELL~ AND PHILIP W. ASKENASE
From the Section of Clinical Immunology and Allergy, Department of Medicine, Yale University School of
Medicine, New Haven, Connecticut 06510
Delayed-type hypersensitivity reactions are mediated by specifically sensitized T
cells that recruit auxiliary effector cells via release of lymphokines (1). Classical
tuberculin-type delayed hypersensitivity (DH)1 reactions are induced when an antigen
is administered in an adjuvant consisting of a water-in-oil emulsion containing
mycobacteria (complete Freund's adjuvant [CFA]) (2). However, reactions with a
delayed time-course can also be elicited in guinea pigs immunized with protein
antigens administered in saline or water-in-oil emulsion not containing mycobacteria.
These reactions differ from D H by their lack of induration and their early disappearance after immunization and testing (3).These evanescent delayed reactions that
occur in animals immunized without mycobacterial adjuvants contain masses of
basophils and are called cutaneous basophil hypersensitivity (CBH), a term that
allows differentiation from D H reactions that contain few basophils (4). CBH reactions
are "carrier specific" and are mediated by sensitized T cells (5-8), but immune serum
can also mediate cutaneous basophil responses (9, 10). These latter reactions are
hapten specific and can be transferred with small amounts of guinea pig IgG1
antibody (11). Thus, both T and B cell-derived factors participate in the generation
of delayed basophil-rich cutaneous hypersensitivity responses.
The fact that T cells can mediate both D H and CBH suggests that the two reactions
are due to separate T cell subsets or that a common T cell is responsible for both
reactions but that the final resulting components of the response (such as basophil
content) are subject to regulatory factors. This latter possibility is suggested by the
fact that guinea pigs immunized with CFA and skin tested at 3-4 wk have classical
D H reactions with few basophils present, but recipients of T cells from these animals
have delayed skin test reactions containing large basophil infiltrates (7). Thus, animals
immunized for basophil-poor classical tuberculin reactions have T cells that can
mediate CBH. The CBH activity of these cells appears to be suppressed, and it has
been proposed that transfer removes them from a suppressive influence, thus allowing
* Supported in part by grants AI-12211, AI-11077, and AI-17555 from the United States Public Health
Service, National Institutes of Health, Bethesda, Maryland.
:~Supported in part by a postdoctoralfellowshipfrom the DermatologyFoundation of America.
t Abbreviations used in this paper." CBH, cutaneous basophil hypersensitivity;CFA, complete Freund's
adjuvant; DEAE, diethylaminoethyl; DH, tuberculin-type delayed hypersensitivity; HBSS, Hanks'
bufferedsalt solution; Ox-KLH, oxazolone-keyholelimpet hemocyanin;Ox-SRBC,oxazolone-sheeperythrocytes; PBS, phosphate-bufferedsaline; PEC, peritoneal exudate cells; Pic-HSA, picryl-human serum
albumin.
J. ExP.MED.© The RockefellerUniversityPress • 0022-1007/82/07/0t 59/14 $ [.00
Volume 156 July 1982 159-172
159
160
CUTANEOUS BASOPHIL HYPERSENSITIVITY SUPPRESSION
t h e m to express C B H . T h e n a t u r e of such a suppressive influence is not known. T h e
c u r r e n t s t u d y reports t h a t when serum a n d p e r i t o n e a l e x u d a t e cells (PEC) from
g u i n e a pigs i m m u n i z e d with C F A are co-transferred i n t r a v e n o u s l y to n o r m a l recipients, the c e l l - m e d i a t e d transfer o f C B H is suppressed. T h e responsible serum factor is
n o n a n t i g e n specific, has a m o l e c u l a r weight of - 7 0 , 0 0 0 , a n d acts p r e f e r e n t i a l l y on
cells from donors t h a t express b a s o p h i l - p o o r D H .
Materials and Methods
Animals. Hartley strain guinea pigs were obtained from Charles River Breeding Laboratories, Inc., Wilmington, MA. Female animals weighing from 250-300 g were used.
Immunization. Guinea pigs were immunized by footpad injection (0.1 ml X 4) using haptenprotein conjugate antigens (100 #g/animal) emulsified with CFA (H37 Ra; Difco Laboratories,
Detroit, MI) with an added 3 mg/ml of ground Mycobacterium tuberculosis. The CFA emulsions
contained either oxazolone coupled to keyhole limpet hemocyanin (Ox-KLH) or Picryl-human
serum albumin (Pic-HSA) obtained and prepared as described previously (7, 10, 11).
Skin Testing. Intradermal skin testing was performed in the shaved flank by injecting soluble
protein antigens in 0.1 ml phosphate-buffered saline (PBS; I0 mM potassium phosphatebuffered 0.15 M NaC1, pH 7.4). Macroscopic reactions were read at 4, 24, and 48 h by
estimating the diameter and extent of erythema and induration. Animals were skin tested with
the antigen to which the donor had been immunized (Ox-KLH and PPD or Pic-HSA and
PPD).
Quantitative Histology. After 48 h, animals were killed by ether anesthesia, and flank skins
were removed and placed in baths of freshly prepared Helly's fixative (Zenker-Formal; Fisher
Scientific Co., Pittsburgh, PA) at room temperature. 24 h later, tissue blocks (2 mm X 10 mm)
were excised from the center of skin reactions, washed with frequent changes of tap water, and
stored in 70% ethanol. Subsequently, they were embedded in paraffin and 4-p.m thick sections
were cut and Giemsa stained at optimum conditions for identification of basophils (10).
Differential cell counts were made in five adjacent central 180 #m Diam oil objective (X 1,000)
fields in the uppermost dermis (10).
Harvesting Cells and Serum. PEC were obtained by the intraperitonea] injection of donors
with 30 ml of sterile light mineral oil (Markol 52; Exxon Corp., Linden, NJ). This was usually
done on day 25 post-immunization, except in those experiments where cells were used early
after immunization (day 7) when it was done on day 4. 3 d later, cells were harvested with
Hanks' buffered salt solution (HBSS), drained from the peritoneal cavity into 250 ml sterile
siliconized centrifuged bottles, and washed three times with HBSS and then resuspended in
RPMI media 1640 with added penicillin (100 U/ml) and streptomycin (100 #g/ml) and 10%
fetal calf serum (Gibco Laboratories, Grand Island Biological Co., Grand Island, NY) at 4°C.
Blood was obtained from the same animals by intracardiac needle puncture under ether
anesthesia before cell harvesting. Serum was separated and used without storage.
Intravenous Transfers. Serum transfer was performed by intravenous injection using the dorsal
foot veins. A volume of 5 ml was administered to each recipient. Cell transfer was also performed
intravenously. This was done after serum injection in cases where both were administered.
Approximately 175-200 x 106 PEC was given to each recipient, with a donor:recipient ratio of
1: 1. The transferred cells were at least 85% viable using the Eosin dye exclusion technique.
Experimental Protocol (Fig. 1). Donors were sensitized with antigens in CFA. On day 28 they
were either skin tested or were killed for harvest of serum and PEC for intravenous transfer.
Normal recipients were skin tested 1-2 h later. After 48 h, macroscopic skin reactions were read,
and animals were killed for fixation and processing of delayed skin reaction sites for quantitation
of the cellular infiltrate.
Affinity Chromatography. Ox-KLH (40 rag) was conjugated to 25 g of Sepharose 2B (Pharmacia
Fine Chemicals, Div. of Pharmacia Inc., Piscataway, NJ) that had been activated previously
with cyanogen bromide (12). A 32-ml column of Ox-KLH Sepharose was calibrated, and 16 ml
of Ox-KLH plus CFA serum was applied, followed by PBS containing 0.02% sodium azide
(Fisher Scientific Co.). The rate of flow was 5 ml/h. The serum that passed through the column
E. BRUCE MITCHELL AND PHILIP W. ASKENASE
Hartley female
guinea pigs
161
= [" Ox-KLHI001R "]
L + CFA
J
25 days
Peritoneal injection of mineral oil
3 clays
1
Skin test (PPD~ OX-KLH)
No skin test
Harvest and transfer
,
PEC (l:l)
Measure macroscopic er~Jthema
(at 24 and 48 hours) and
microscopic basophils 148hours)
[
I
Serum15ml)
PEC +serum
Skin test recipients IPPD, Ox-KLH)
Measure macroscopic erythema 124and 48 hours)
and microscopic basophils 148 hours)
FIG. 1. Experimental protocol. Donors were sensitized with antigens in CFA. On day 28, they
were either skin tested or were killed for harvest of serum and PEC for intravenous transfer. Normal
recipients were skin tested 1-2 h later. After 48 h, macroscopic skin reactions were read, and animals
were killed for fixation and processing of delayed reaction sites for quantitation of the cellular
infiltrate.
became diluted to 25 ml and was concentrated back to 16 ml using negative pressure. T h e n the
serum was dialyzed overnight against sterile PBS and was then transferred.
1on-Exchange Chromatography. Serum globulins were precipitated using 18 g% sodium sulphate
(Na2SO4). T h e precipitate was dissolved with PBS and dialyzed for 3 d against 0.01 M
potassium phosphate buffer, p H 8.0 (starting buffer). T h e solution was centrifuged at 10,000 g
for 10 min to remove debris, and the supernatant was applied by gravity to a 10- X 2.5-cm
column containing diethylaminoethyl (DEAE) cellulose 0Nhatman, Maidstone, Kent, U. K.)
that had been equilibrated previously with the starting buffer. Fractions were collected every
5 min at a flow rate of 30 m l / h . IgG2 eluted with the starting buffer and then IgG1 eluted with
a second buffer (0.04 M phosphate buffer, p H 6.3). The third buffer (0.5 M NaC1) eluted the
other proteins (11). Nominal protein concentrations in all samples were calculated on the basis
that each 1 m g / m l had an absorbance at 280 nm of 1.3 (the extinction coefficient of IgG).
Correction factors were used to account for dilution or concentration of the fraction pools when
compared with the original serum. All samples were dialyzed overnight against sterile PBS and
were then transferred.
Gel Filtration Chromatography. A column 5.0 × 100 cm was filled with Sephadex G-200
(Pharmacia Fine Chemicals, Div. of Pharmacia Inc.). After calibration, 19 ml of fresh immune
serum was applied by gravity, followed by PBS containing 0.2% azide and 5 m M E D T A at
4°C. The column was run over 4 d by upward flow at a pressure of 10 cm and a rate of 28 m l /
h, with fractions collected every 15 min. The absorbance at 280 nm was determined for each
fraction and three peaks were pooled and processed as stated above and used immediately.
Titration of Hemagglutinating Antibodies. Coupling of oxazolone to sheep erythrocytes (SRBC)
and hemagglutination was performed as described previously (13). Duplicate serial twofold
dilutions of each sample were made in PBS containing 2% heat-inactivated normal rabbit
serum, absorbed previously with O x - S R B C (13). T h e n 25/d of 0.25% vol/vol O x - S R B C in the
same buffer were added to each 25-/~1 dilution in conical-bottomed microtiter trays. The trays
were incubated for 1 h at room temperature and then overnight at 4°C. The titer was read as
the last well showing definite agglutination. For a n t i - K L H titers, glutaraldehyde was used to
couple K L H to S R B C (14).
Passive Cutaneous Anaphylaxis Titration of Antibody. Antisera (0.1 ml) in various dilutions were
injected intradermally into 250-g normal guinea pigs. 3-4 h later, 1 mg antigen i n 1 ml of 1%
Evans blue dye was injected intravenously. The sites were read 20 min later for the diameter of
extravasated dye.
162
CUTANEOUS BASOPHIL HYPERSENSITIVITY SUPPRESSION
Statistical Analysis. A n u n p a i r e d t test w a s u s e d to c o m p u t e t h e s i g n i f i c a n c e (P < 0.05) o f
differences between groups.
Results
Cell Transfer of CB~I from Guinea Pigs Immunizedfor DH (Fig. 2).
PEC from donors
immunized with Ox-KLH plus CFA 28 d previously was administered intravenously
to recipients and transferred the ability to elicit delayed responses to the immunizing
antigen Ox-KLH (Fig. 2 a). Macroscopically, the reaction size in the recipients was
significantly smaller (P < 0.001) than those elicited in the donors. As reported
previously (7), skin reactions elicited with Ox-KLH in the recipients were basophil
rich (Fig. 2 a), unlike the DH responses of the donors that were basophil poor (P <
0.001). Fig. 2 b shows that PPD skin testing yielded results comparable to those seen
with the immunizing hapten protein conjugate. The reactions elicited in cell transfer
recipients were smaller than those of the actively immunized (P < 0.001) and
contained significantly increased numbers of basophils (P < 0.001). These results
suggest that (a) cells are able to transfer delayed responses but are insufficient to
induce DH and instead induce reactions resembling CBH, and (b) actively immunized
animals seem to possess the ability to inhibit cell-mediated CBH.
Immune Serum Modulates Macroscopicand Microscopic Aspects of Delayed Skin Test Responses
a Ox-KLH skin tests
a~
P < 0.001
30
-150
4.1
u4
E
2o-
~
x
10-
-50
._~
E
,o
0
No.of animals
12 12
18 18
12 12
Immunization
Active
Passive
None
b PPD skin tests
÷l
30-
- 150
P < O.001
,,4
~
.~
-100 ~x
Z0-
-50
~" 10-
~
E
0
No.of animals 12 12
18 18
12 12
Immunization
Passive
None
Active
Fro. 2. Macroscopic erythema and microscopic bsaophils in skin test responses of actively sensitized
guinea pigs or recipients of i m m u n e PEC. Donors were immunized with 100 p,g O x - K L H emulsified
with CFA, PEC were harvested at 28 d. Data were pooled from six experiments. Actively immunized
and PEC recipient animals were skin tested with 100 #g O x - K L H (Fig. 2 a) and with 25 p,g PPD
(Fig. 2 b). rn, erythema; ~ , basophils.
163
E. B R U C E M I T C H E L L A N D P H I L I P W. A S K E N A S E
in Recipients of Immune Cells (Fig. 3 and Table I).
When immune PEC and immune
serum that were harvested 28 d after immunization were transferred intravenously,
skin testing with the immunizing conjugate O x - K L H elicited delayed reactions that
were macroscopically larger than those seen in animals that received cells alone (Fig.
3 a). Such augmented responses were evident at 24 and 48 h (only 48-h responses are
shown). Early (4 h) indurated and hemorrhagic lesions (i.e., Arthus-like) were not
seen in recipients. I m m u n e serum alone transferred the ability to elicit skin test
reactions to the immunizing conjugate that were smaller than those seen with cells.
P P D responses were less, though significantly (P < 0.001) augmented by concomitant
administration of PEC and serum, whereas immune serum alone did not have the
ability to transfer reactivity to P P D skin testing (Fig. 3 b).
Concomitant transfer of i m m u n e serum with immune cells altered the histological
nature of the elicited skin test reactions. Individually, cells and serum allowed the
elicitation of respectively strong and weak cutaneous basophil responses. Together,
their administration resulted in suppression of the strong basophil infiltrate that
occurred with cells alone. The suppression of basophil infiltration was evident to skin
testing with the immunizing conjugate O x - K L H (Fig. 3 a) and P P D (Fig. 3 b). The
reduced basophil response is all the more surprising when viewed in the light of the
a Ox-KLH skin tests
P < 0.0Ol
30-
- 150
"t'1
0.001
.H
-1~
~,~ lo-
-50
2
E
0
o
No. of animals
18 18
6
6
18 18
12 12
12 12
Transferred
Cells
alone
Serum
alone
Cells +
rActive . ]
None
serum
L immunlz..I
b PPD skin tests
+1
- 1~0 _~
30<6
20-
- 1~
P < 0.001
P < 0.001
~" 10-
INII
0
No.ofanimals
18 18
12 12
18 18
Transferred
Cells
alone
Serum
alone
Cells
+
serum
x
3a
12 12
12 12
[ Active ]
immuniz.j
None
-5o
N
o
.~
Lo
-~
E
Fro. 3. I m m u n e serum m o d u l a t e s skin test responses in recipients o f i m m u n e PEC. Donors were
i m m u n i z e d w i t h 100 p,g O x - K L H emulsified with CFA. PEC (1 donor: 1 recipient) a n d i m m u n e
serum (5 ml) were harvested at 28 d a n d transferred intravenously. D a t a were pooled from six
experiments. Actively i m m u n i z e d and recipient a n i m a l s were skin tested with 100 ~g O x - K L H (Fig.
3 a) a n d with 25/~g P P D (Fig. 3 b). Statistically significant differences represent comparisons to the
group that received cells alone, r-n, erythema; r~, basophils.
164
CUTANEOUS BASOPHIL HYPERSENSITIVITY SUPPRESSION
TABLE I
Immune Serum Modulates Skin Test Responses to Various Doses of Antigen in Recipients of Immune PEC*
Recipient's skin test responses
Transferred
Cells(PEC)
Serum
Cells + serum
Nil
Number of
animals
Skin test
dose
Macroscopic erythema
Microscopic cells per 5 1,000× fields ± SE
Basophils
Eosinophils
Neutrophils
Mononuclear
102±24
134± 12
139 ± 30
11±4
7.4 1
13 ± 3
3± 1
12±5
5 :l: 3
74± 11
54± 7
67 ± 20
34±3
37+7
66± 7
~tg
mm ± SE
6
18
6
30
100
300
9± 1
14± 1
16 .4 2.0
6
6
6
30
100
300
3± I
9±4
13± 1
17±8
29±6
24± 7
7± 1
19+6
17±3
12±4
4±2
11 ± 3
6
18
6
30
100
300
16 ± 2:~
27 :t: 2:]:
32 ± 4:~
49 ± 10~:
54 :i: 10:[:
33± 10:[:
13 ± 4
18 ± 4
18±4
10 ± 3
30 ± 12~
87 ± 20:[:
6
6
6
30
100
300
0
2±1
2:t:2
6-43
2±1
5±3
6-43
6:t:3
4-42
1+I
8+6
10-46
55 ± 17
79 :i: 4:~
191 ± 11~:
43-43
34±3
72±14
* Donors were immunized with 100 #g Ox-KLH emulsified with CFA. PEC and immune serum harvested
at 28 d were transferred intravenously at a dose of 1 donor to 1 recipient for the PEC and 5 ml for the
serum. 1-2 h after transfer, recipient animals were skin tested with 0.1 ml PBS containing 300/~g, I00
/~g, and 30 #g of Ox-KLH.
:]: Statistically significant (P < 0.05) when compared with the group that received cells alone.
a u g m e n t e d m a c r o s c o p i c r e a c t i o n s t h a t w e r e p r e s e n t w h e n skin t e s t i n g w i t h t h e
c o n j u g a t e in p a r t i c u l a r (Fig. 3 a). T h e r e w e r e m o r e n e u t r o p h i l s a n d m o n o n u c l e a r cells
in r e a c t i o n s o f a n i m a l s t h a t r e c e i v e d cells plus s e r u m t h a n in those t h a t r e c e i v e d cells
a l o n e , a n d s e r u m a l o n e d i d n o t t r a n s f e r t h e a b i l i t y to elicit skin r e a c t i o n s t h a t h a d
h i g h n e u t r o p h i l c o u n t s ( T a b l e I). T h u s , r e c i p i e n t s o f cells p l u s s e r u m h a d s t r o n g l y
e r y t h e m a t o u s a n d i n d u r a t e d r e a c t i o n s t h a t h a d a p r e d o m i n a n t m o n o n u c l e a r cell
infiltrate, a n e u t r o p h i l c o m p o n e n t , a n d d i m i n i s h e d basophils. I n t h e s e respects,
r e a c t i o n s in r e c i p i e n t s o f cells plus s e r u m r e s e m b l e d d o n o r responses m o r e t h a n
r e a c t i o n s e l i c i t e d in r e c i p i e n t s o f cells alone.
T h e f i n d i n g t h a t i m m u n e s e r u m a u g m e n t e d t h e m a c r o s c o p i c size o f r e a c t i o n s
e l i c i t e d w i t h t h e i m m u n i z i n g c o n j u g a t e in r e c i p i e n t s o f i m m u n e P E C was d e m o n s t r a t e d at a w i d e r a n g e o f skin test doses, 30 #g, 100/~g, a n d 300/~g, as was s u p p r e s s i o n
o f b a s o p h i l i n f i l t r a t i o n ( T a b l e I). T h u s , i m m u n e s e r u m m o d u l a t i o n o f t h e m a c r o s c o p i c
a n d m i c r o s c o p i c aspects o f c e l l - d e p e n d e n t r e a c t i o n s was n o t d e p e n d e n t o n a n y
p a r t i c u l a r skin test dose.
C o n c o m i t a n t t r a n s f e r o f i m m u n e cells (I:1 ratio) a n d f o u r f o l d d i l u t i o n s o f s e r u m
revealed that significnt macroscopic augmentation and microscopic basophil suppression was n o t p r e s e n t at a v o l u m e o f 1.25 m l or less. N o r m a l s e r u m (5 ml) d i d n o t
possess e i t h e r a c t i v i t y ( d a t a n o t shown).
Macroscopic Augmentation Is Antigen Specific, and Microscopic Suppression of Basophil
Infiltrates Is Nonspecific (Fig. 4 and Table II). T o a s c e r t a i n w h e t h e r t h e a b i l i t y o f
165
E. BRUCE M I T C H E L L AND PHILIP W. ASKENASE
Ox-KLH skin tests
30-
Jl
4-t
P < 0.001
- 150
r
+~
20-
~.,
o~ 10-
-1(~
,O0
~1
- 50
.~
0o
No. of animals
18
Cells
Serum
Transferred:
18
Ox KLH
18
18
6
6
6
0x KLH
0x KLH
Ox KLH
pic HSA
6
Ox KLH
3 3
pic HSA
b Pic-HSA skin tests
40-
-I-i
.g
44
NS
30-
-
150
- ]~
20-
.-X
e
E
~
-50 ~
10-
0
No.of animals
3
Transferred: Cells
3
pic HSA
Serum
3 3
3 3
6 3
3 3
pic HSA
pic HSA
pic HSA
Ox KLH
pic HSA
Ox KLH
FIO. 4. Specificity of immune serum that modulates skin test responses in recipients of immune
PEC. Separate groups of donors were immunized with either Ox-KLH or Pic-HSA (t00/~g each)
emulsified with CFA. PEC and immune serum were harvested at 28 d. Animals received immune
cells alone, immune cells plus homologous immune serum, or immune cells plus heterologous
immune serum. Animals were skin tested with 100 #g Ox-KLH (Fig. 4a) or with 100 #g Pic-HSA
(Fig. 4b). D, erythema; ~ , basophils.
TABLE II
Antigen-Affinity Chromatography of Immune Serum That Modulates Skin Test
Responses in Recipients of Immune Cells*
Recipient's skin test responses
I m m u n e components transferred
Macroscopic
erythema
Microscopic
basophils per 5
1,000× fields
± SE
mm ± SE
Cells alone
Cells + serum
Cells + O x - K L H Sepharose
column-passed serum
12.3 ± 1.2
21.0 ± 1.5:~
t3.7 ± 0.3
87.7 ± 4.5
53.3 ± 3.5:~
46.3 ± 11.3:~
* Donors were immunized with 100 #g O x - K L H emulsified with CFA. PEC
and immune serum were harvested at 28 d. Serum was passed through an
Ox-KLH Sepharose affinity column. Recipient animals were skin tested with
100 #g Ox-KLH. Each group consisted of three animals.
:l: Statistically significant (P < 0.05) when compared with cells alone group.
166
CUTANEOUS BASOPHIL HYPERSENSITIVITY SUPPRESSION
immune serum to macroscopically augment reaction size and suppress basophil
infiltration was antigen specific, two groups of animals were immunized, each with a
different hapten-protein conjugate in CFA. O x - K L H and Pic-HSA were used as the
immunizing antigens. Crossover studies were done, whereby immune cells of one
specificity were transferred either alone with homologous immune serum or with
heterologous immune serum.
Fig. 4 a shows that macroscopic augmentation of elicited reactions was antigen
specific. Pic-HSA plus CFA immune serum did not significantly enlarge O x - K L H
responses elicited after transfer of cells with O x - K L H specificity, whereas O x - K L H
plus CFA immune serum did. In contrast, both homologous and heterologous immune
CFA serum had the ability, when administered with O x - K L H cells, of suppressing
basophil infiltration. Fig. 4 b shows that the heterologous Pic-HSA plus CFA serum
that nonspecifically suppressed the ability of immune cells with O x - K L H specificity
to transfer the elicitability of basophil-rich delayed reactions could suppress basophil
infiltration in a homologous system when administeed with cells of Pic-HSA specificity. Also, O x - K L H plus CFA-immune serum transferred with immune cells of PicHSA specificity suppressed the infiltration of basophils into Pic-HSA skin test lesions
(Fig. 4 b). Macroscopically, homologous Pic-HSA-immune cells plus immune serum
did augment reaction size, but this augmentation did not reach statistical significance.
In contrast, O x - K L H plus CFA serum had a much reduced effect upon the macroscopic size of Pic-HSA skin tests when given in combination with Pic-HSA-immune
cells.
These data suggest that the ability of i m m u n e CFA serum to macroscopically
augment the elicited skin reaction size in recipients of immune cells is antigen specific,
as it is only significant in a homologous system. However, immune CFA serum can
suppress the infiltration of basophils into such reactions in an antigen-nonspecific
manner, as evidenced in two different immunization protocols.
An antigen affinity column was used to further investigate the dual activity of
immune CFA serum. 16 ml of 28-d O x - K L H plus CFA-immune serum was passed
through an Ox-KLH-Sepharose column. All anti-oxazolone and a n t i - K L H hemagglutinating antibody was removed (data not shown). The passed serum was concentrated and administered in 5-ml equivalent volumes. Table II shows that immune
serum that macroscopically augmented reactions elicited in i m m m u n e cell recipients
lost this activity after passage through an antigen affinity column. In contrast, the
ability of the immune serum to suppress basophil infiltration into such reactions was
not altered by passage through an antigen affinity column. This further suggests that
these two serum activities are separable and distinct, as one (macroscopic augmentation) binds to an antigen column and one (basophil suppression) does not.
Fractionation of Immune Serum That Modulates Skin Test Responses in Recipients of Immune
Cells (Tables III and IV). An 18 g% Na2SO4 glolubin fraction of immune serum (OxK L H plus CFA) was applied to a DEAE column and eluted as three peaks with
stepwise changes in buffer. Hemagglutination titers, PCA titers, and macroscopic
augmenting and basophil-suppressing activity were assessed for each fraction. The
initial globulin fraction retained the ability to both macroscopically augment reactions
and suppress the infiltration of basophils when administered with immune PEC to
recipient animals that were skin tested with O x - K L H . The IgG2-containing fraction
retained the ability of the whole serum to augment macroscopic reaction size under
E. BRUCE MITCHELL AND PHILIP W. ASKENASE
167
TABLE III
DEAE Ion Exchange Chromatography of Immune Serum That Modulates Skin
Test Responses in Recipients of Immune Cells*
Reclpient's skin test responses
Immune components
transferred
Macroscopic
erythema
Microscopic basophils
per 5 1,000× fields 4SE
ram ± SE
Cells alone
Cells + serum
Cells + IgG2 fraction
13.0 4- 0.9
27.3 4- 1.3:1:
18.2 4- 1.3~
146.8 4- 12.0
47.0 4- 16.5~
137.2 4- 13.2
* Donors were immunized with 100 pg Ox-KLH emulsified with CFA. PEC
and immune serum were harvested at 28 d. Serum was fractionated on a
DEAE cellulose column. Data is shown only for the IgG¢ fraction. Recipient
animals were skin tested with 100/~g Ox-KLH. Results were pooled from
two experiments with six animals in each group.
:[: Statistically significant when compared with the cells alone group.
TABLE IV
Sephadex G200 Gel Filtration Chromatography of Immune Serum That
Modulates Skin Test Responses in Recipients of Immune Cells*
Recipient's skin test responses
Immune components transferred
Macroscopic
erythema
Microscopic
basophils per
5 1,000X
fields 4- SE
mm ± SE
Cells alone
Cells + excluded fraction
Cells + IgG-sized fraction
Ceils + albumin-sized fraction
11.2 ± 0.5
10.0 4- 1.2
18.7 ± 1.8~
19.0 4- 2.52:~
112.8 4- 6.3
112.0 ± 24.6
127.0 + 10.7
51.0 ± 7.1~
* Donors were immunized with 100/~g Ox-KLH emulsified with CFA. PEC
and immune serum were harvested at 28 d. Serum was fractionated on a
Sephadex G-200 column. Recipient animals were skin tested with 100 #g
Ox-KLH. Each group consisted of three animals.
:~ Statistically significant when compared with the cells alone group.
s i m i l a r c i r c u m s t a n c e s , b u t this f r a c t i o n d i d n o t h a v e t h e b a s o p h i l - s u p p r e s s i n g a c t i v i t y
( T a b l e III). N e i t h e r t h e IgG1 s e r u m f r a c t i o n n o r t h e t h i r d f r a c t i o n f r o m t h e D E A E
column had macroscopic augmenting activity or detectable basophil-suppressing
a c t i v i t y ; this l a t t e r a c t i v i t y m i g h t h a v e b e e n lost d u r i n g f r a c t i o n a t i o n . T h e IgG2
fraction had most of the hemagglutinating anti-oxazolone and anti-KLH antibody
a c t i v i t y o f w h o l e s e r u m , a n d t h e IgG1 f r a c t i o n h a d v i r t u a l l y all o f t h e P C A a c t i v i t y o f
b o t h specificities ( d a t a n o t shown).
A S e p h a d e x G - 2 0 0 c o l u m n (90 X 5 c m ) was used to assess t h e size o f s e r u m factors
t h a t h a d t h e a b i l i t y to s u p p r e s s b a s o p h i l infiltrates. 19 m l o f i m m u n e s e r u m was
a p p l i e d to t h e c o l u m n , a n d t h r e e m a j o r p r o t e i n p e a k s w e r e c o l l e c t e d . C o n c e n t r a t e d
pools o f f r a c t i o n s r e p r e s e n t i n g t h e s e t h r e e p e a k s w e r e t r a n s f e r r e d c o n c o m i t a n t l y w i t h
i m m u n e cells. T a b l e I V shows t h a t n e i t h e r t h e e x c l u d e d f r a c t i o n n o r t h e I g G - s i z e d
f r a c t i o n h a d t h e a b i l i t y to s u p p r e s s b a s o p h i l i n f l u x to t h e local r e a c t i o n site. T h e
168
CUTANEOUS BASOPHIL HYPERSENSITIVITY SUPPRESSION
TABLE V
Comparison of 7-d vs. 28-d Immune Serum Activity in Modulation of Skin
Test Responses in Recipients of 7-d Immune Cells *
Recipient's skin test responses
Immune components
transferred
Cells (7 d)
Cells (7 d) + serum (7 d)
Cells (7 d) + serum (28 d)
Macroscopic
erythema
Microscopic basophils per 5 1,000×
fields 4" SE
rnm + SE
13 ± 2.3
11 ± 3.0
17 ± 1.2
134 + 33.2
156 ± 51.6
115 ± 12.5
* Donors were immunized with 100/~g Ox-KLH emulsified with CFA. PEC
and immune serum were harvested at either 7 d or 28 d. Early (7 d) cells
were administered with either early (7 d) serum or late (28 d) serum.
Recipient animals were skin tested with 100/lg Ox-KLH. There were three
animals in each group. Later (28 d) serum had the property of significantly
suppressing basophil infiltration when given with late (28 d) cells.
a l b u m i n - s i z e d fraction d i d have this activity, thus suggesting t h a t the responsible
serum factor has an ~ 70,000 mol wt. Both the IgG-sized a n d a l b u m i n - s i z e d fractions
h a d macroscopic a u g m e n t i n g activity, suggesting t h a t some o v e r l a p o f these peaks
occurred. T h i s m i g h t be because the c o l u m n was o v e r l o a d e d (19 ml o f serum was
a p p l i e d , a n d fraction pools were transferred to three recipients) in a n t i c i p a t i o n o f
losses in b a s o p h i l suppression activity.
Modulation of Skin Reactions Is a Property of Late (28-d) Serum plus Cells and Not of Either
Early (7-d) Serum or Cells (Table V). A n i m a l s i m m u n i z e d to express D H responses
will express b a s o p h i l - c o n t a i n i n g responses 7 d after i m m u n i z a t i o n (7, 10, 15). T h e r e fore, the effects o f c o n c o m i t a n t transfer o f 7-d i m m u n e cells plus either 7-d or 28-d
i m m u n e s e r u m were investigated. T a b l e V shows t h a t n e i t h e r 7-d nor 28-d serum h a d
the a b i l i t y to suppress the b a s o p h i l infiltration o f reactions elicited after transfer of 7d cells. Some m a c r o s c o p i c a u g m e n t a t i o n d i d occur with 28-d serum, t h o u g h this was
not statistically significant. T h e s e results suggest t h a t i m m u n e cells g e n e r a t e d early
(7 d) after i m m u n i z a t i o n for D H responses are different from l a t e r (28-d) o c c u r r i n g
i m m u n e cells because they are m o r e difficult to m a c r o s c o p i c a l l y a u g m e n t a n d because
they are not susceptible to b a s o p h i l suppression b y late (28-d) i m m u n e serum k n o w n
to have such properties. By inference, the d a t a suggest t h a t early cells are m o r e likely
to express the c a p a c i t y to recruit hasophils to d e l a y e d reactions as is the case.
Discussion
D e l a y e d c u t a n e o u s reactions t h a t are elicited b y specific a n t i g e n in a sensitized host
are c o m p l e x processes consisting of several components. W h e n g u i n e a pigs are
i m m u n i z e d with a p r o t e i n a n t i g e n emulsified with c o m p l e t e F r e u n d ' s a d j u v a n t ,
classical t u b e r c u l i n - t y p e D H reactions are elicited b y P P D or b y the i m m u n i z i n g
p r o t e i n antigen. M a c r o s c o p i c a l l y , D H reactions have a characteristic d e l a y e d onset
(6-8 h after testing), a b r o a d p e a k over 24-48 h, a n d are c h a r a c t e r i z e d by strong
e r y t h e m a a n d i n d u r a t i o n . Microscopically, D H reactions are rich in m o n o c y t e /
m a c r o p h a g e s t h a t are r e c r u i t e d from the bone m a r r o w via the blood. Basophils
comprise < 5 % o f the infiltrate o f these classical D H reactions. In contrast, basophils
E. BRUCE MITCHELL AND PHILIP W. ASKENASE
169
comprise 25-60% of the infiltrate in delayed, weakly erythematous, and nonindurated
CBH responses that are elicited in guinea pigs immunized with proteins administered
without mycobacterial adjuvant (4, 7, 15).
The T cell dependence of D H responses is apparent from the hapten-carrier
specificity of reactions elicited in actively sensitized animals and by the fact that
elicitability of delayed reactions of similar fine specifity requirements is transferable
from these animals to normal recipients by live sensitized T cells and not by soluble
serum components (7, 16). However, careful examination of reactions elicited in
recipients of immune T cells reveals that these responses differ significantly from those
of the donors. In particular, recipient responses are weaker in intensity of erythema
and induration, and microscopically they contain significant infiltrates of basophils
(7). Therefore, recipient reactions resemble CBH responses rather than D H responses
that are elicited in the donors. Thus, the landmark cell transfer experiments of
Landsteiner and Chase in 1942 were in fact probably T cell transfers of CBH from
donors with DH. These T cell transfers of CBH from donors with D H are not due to
major histocompatibility complex restrictions or an allogeneic effect, as they occur
between strain 13 inbred guinea pigs (7) as well as between outbred Hartley guinea
pigs.
The current study has compared reactions elicited in recipients of cells alone to
those of animals receiving both immune cells and immune serum. Our findings
indicate that components of immune serum act in concert with immune T cells to
produce elicitation of reactions in recipients that more fully resemble those of the
donors then do reactions in recipients of cells alone. At least two serum components
are responsible for this modulation of T cell-dependent delayed reactions. One is an
antigen-specific IgG2 antibody that leads to macroscopic augmentation of the cellmediated response. The other is a nonantigen-specific albumin-sized serum component
that leads to suppression of the T cell-dependent microscopic basophil infiltrates.
The macroscopic augmentation was antigen specific, as it was not present with
serum from donors immunized with another antigen in CFA and was removed by an
antigen affinity column. Asherson and Loewi (17) also reported a synergistic action of
immune cells and immune serum in the passive transfer of delayed hypersensitivity.
However, partial augmentation of cell transfer could be demonstrated after the
induction or an irrelevant antigen-antibody reaction at the skin test site (18, 19). The
current experiments support the concept that an antigen-antibody reaction is responsible for macroscopic augmentation by demonstrating that IgG~ antibody has this
property. This antibody isotype has the ability to fix complement, and such a
mechanism or antigen retention at the skin test site might participate in the augmented
response. However, the exact mechanism of augmentation by antibody is not clear, as
there is differing evidence available for different antigens and immunization protocols
(18, 20, 21). Only a small but significant increase in macroscopic reactions was
demonstrable when skin testing with PPD, whereas a more marked augmentation
was present when testing with the hapten-protein conjugate. A poor IgG antibody
response to PPD at 4 wk after immunization might explain this difference.
Immune serum from animals immunized with CFA was found to have the very
important property of suppressing the basophil-rich histology present after transfer of
immune ceils alone. Interestingly, this property was not found in the IgG2 serum
fraction and thus could be differentiated from the macroscopic augmenting ability.
170
C U T A N E O U S BASOPHIL HYPERSENSITIVITY SUPPRESSION
The activity of suppressing basophils was not antigen specific, as it could be elicited
when immune ceils were transferred in combination with serum from donors immunized with another antigen in CFA, and basophil-suppressing activity was still present
after specific immune serum was passed through an antigen affinity column. Gel
filtration chromatography revealed that the basophil-suppressing activity resided in
the albumin-sized fraction, though possibly it was caused by an even smaller molecule
that was albumin bound. It is tempting to speculate that the activity was caused by
a cell product and to compare it with analogous regulatory factors in CFA-induced
serum that selectively suppress IgE antibody responses (22). In the mouse, Kishimoto
(23) has demonstrated that such a factor is a T cell product with a 55,000-60,000 mol
wt. Thus far, our attempts at producing the basophil-suppressing factor in vitro have
not been successful (Mitchell and Askenase, unpublished observations).
Basophil-rich delayed-onset reactions are elicited at early intervals after immunization with soluble protein antigens emulsified with CFA, but, at later intervals,
basophil-poor and mononuclear cell-rich D H reactions are elicited (4, 15). Comparison
of reactions elicited after transfer only of early cells (7-d postimmunization) with those
elicited after transfer of early cells plus early serum revealed that macroscopic
augmentation and basophil suppression did not occur. Later serum (28 d), which was
known to possess basophil-suppressing activity when administered with late (28 d)
cells, failed to suppress cell-dependent basophil infiltrates when transferred with early
(7 d) ceils. These results suggest a distinct difference between early and late immune
cells after immunization with CFA. Early ceils are resistant to CBH-suppressive
activity of late serum, and it is at this stage that early cells are most effective in
recruiting basophils. It has been shown (24) that cells from animals immunized with
CFA in which hospital infiltrates are suppressed are potent producers of lymphokines
that are chemotactic for basophils. The serum activity we described might either
prevent the production of, or render inoperative, such chemotactic stimuli.
In conclusion, our results suggest that elicitibility of D H reactions is not achieved
by transfer o f T cells alone, although these responses are T cell dependent. Additional
antigen-specific and antigen-nonspecific factors in immune serum are required to
elicit delayed responses in recipients that more faithfully reproduce those elicited in
the donors. The biological utility of this phenomenon is not clear. However, delayed
basophil infiltrates are required for immune rejection of some large multicellular
parasites, 2 whereas monocyte/macrophage-rich responses are more appropriate to
immune resistance to facultative intracellular microorganisms, such as mycobacteria
(1, 25, 26). Therefore, the ability of specific and nonspecific factors in immune serum
to modulate the basophil vs. monocyte/macrophage component of T cell-dependent
tissue responses might have important biological consequences.
Summary
Guinea pigs immunized with protein antigens emulsified with complete Freund's
adjuvant (CFA) and skin tested at 3-4 wk have classical tuberculin-type delayed
hypersensitivity (DH) reactions with few basophils present. However, recipients of T
cells from these animals have delayed responses containing large basophil infiltrates
2 Brown, S. J., S. J. Galli, G. Gliech, and P. W. Askenase. 1982. Ablation of immunity to Ambyloma
americanium by anti-basophil serum: cooperation between basophils and eosinophils in expression of
immunity to ectoparasites (ticks) in guinea pigs.,]. ImmunoL In press.
E. BRUCE MITCHELL AND PHILIP W. ASKENASE
171
a n d thus resemble basophil-rich cutaneous basophil hypersensitivity (CBH) responses
that are elicited in animals i m m u n i z e d without CFA. This suggests that animals
i m m u n i z e d with C F A have T cells with basophil-recruiting capacity but that this
activity is suppressed.
Using a transfer system, we found that i m m u n e serum from donors i m m u n i z e d
with C F A h a d the ability to suppress the basophil-recruiting capacity of i m m u n e T
cells. W h e n i m m u n e serum and peritoneal exudate cells from guinea pigs i m m u n i z e d
with C F A were co-transferred intravenously to normal recipients, the cell-mediated
transfer of basophil-rich responses was suppressed. T h e responsible serum factor was
antigen nonspecific, had an - 7 0 , 0 0 0 mol wt, a n d acted preferentially on cells from
donors that express basophil-poor D H responses.
Thus, tuberculin-type delayed hypersensitivity and C B H might be mediated by a
c o m m o n T cell, but the resulting basophil c o m p o n e n t o f the delayed response depends
on the modulation of T cell recruitment of basophils by factors in C F A - i m m u n e
serum.
The authors are grateful to Christine Haley, Robin Meade, and Lisa Langlois for their expert
technical assistance and to Ann Criscuolo for her secretarial skills.
Receivedfor publication 4 February 1982 and in revisedform 14 April 1982.
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