Download Experimental Production of Rabbit Anti-Guinea-Pig

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Copyright Cl 1977 by The Williams & Wilk ins Co.
THE J OURNAL OF iNvEsTIGATIVE DERMATOLOGY,
Vol. 68, No.5
Printed in U.S A .
REPORTS
EXPERIMENTAL PRODUCTION OF RABBIT ANTI-GUINEA-PIG
EPIDERMAL CELL SERA. COMPARISON TO PEMPHIGUS ANTIBODIES
MASAHIRO TAK1GAWA , M .D ., AND SADAO IMAMURA , M .D
Department of Dermatology. Faculty of Medicine, Kyoto University . Kyoto. Japan
Anti-epidermal cell sera (AES) were obtained by immunizing rabbits with enzymatically
dispersed, viable guinea-pig epidermal cells followed by absorption with guinea-pig red
blood cells, spleen and thymus cells, and liver powders. Complement-mediated cytotoxicity
and immunofluorescence demonstrated that AES were directed towards cell surface antigens specific for stratified squamous epithelia of guinea pigs, and cross reacted with the
corresponding tissues of humans and monkeys. Immunofluorescence revealed that AES
reacted with Hassall's corpuscles and surrounding epithelial cells of the guinea-pig thymus
which seemed to share common antigens with the epidermis; AES gave no reaction with
other organs. While antigens reactive with pemphigus antibodies (PA) were demonstrated
by membrane immunofluorescence to be present on the epidermal cell surface, PA showed
no cytotoxicity to guinea-pig and human epidermal cells. Re-treatment of isolated epidermal
cells with trypsin showed that antigens ractive with PA were more s usceptible to the enzyme
than those reactive with AES. These findings suggest that the cell surface antigens binding
AES are different from the antigens which bind PA.
Studies of the cell surface structure have revealed that certain kinds of cells possess a variety
of antigens on their membrane. Some of these
antigens seem to be exclusive for a specifi.c population or particular to a certain stage in their differentiation. Such antigens have been well studied in
lymphoid cells I11. Although epidermal tissue-specific antigens have been widely investigated 12-5],
they have rarely been demonstrated on the cell
surface. Recently, the presence of histocompatibility antigens and a new system of alloantigens was
demonstrated on the surface of mouse epidermal
cells [6-8]. Also, heteroantigens were detected on
the epidermal cells of mouse and rat using heterologous antisera; the sera reacted with the surface
antigens of epidermal cells having a broader specificity which cuts across species differences [9,1 01.
There is. therefore, a similarity between heteroloManuscript received May 17, 1976; accepted for publication December 2, 1976.
This work was supported, in part, by a Research
Grant for Specific Disease from the Ministry of Health
and Welfare.
Reprint requests to: Dr. M. Takigawa, Department of
Dermatology , Faculty of Medicine, Kyoto University.
Kyoto 606, Japan.
Abbreviations:
AES: anti-epidermal cell sera
C: complement
FCS: fetal calf serum
FITC: fluorescein isoth.iocyanate
MEM: minimal essential medium
PA: pemphigus antibodies
259
gous antisera and pemphigus antibodies (PA) in
that they are both directed towards the tissuespecific but not species-specific antigens of the epidermis D 1]. This report deals with the experimental production of heterologous antisera to guineapig epidermal cells (AES) and a comparison of the
reactivities to guinea-pig epidermis between AES
and PA.
MATERIALS AND METHODS
Isolation of epidermal cells . A modification of the
methods of Karasek and Charlton [12], and Vaughan
and Bernstein [13] was used. The back, flanks, and
abdomen of out bred guinea pigs (Hartley strain) of both
sexes. weighing 250 to 500 gm. were depilated, shaved.
and cleaned with 70'*' ethyl a lcohol. Thin skin fragments were removed with razors and washed briefly in
Eagle's minimal essential medium (MEM) containing
100 units/ml of penicillin and 100 p.g/ml of streptomycin. The fragments were incubated at 37•c in a 0.3%
solution of trypsin (1:250, Difco) which was prepared in
CAh- and Mg++-free phosphate-buffered saline and
sterilized by membran filtration . The time of incubation was 45 to 75 min depending on the thickness of the
fragments. After incubation the epidermis was separated from the dermis with forceps, cut into about 5-mm
rectangles, and gently stirred magnetically in a flask
containing MEM for about 30 min at. 37•c to disperse
the cells. The resulting cell suspension was filtered
through cotton wool. and the cells were washed 3 times
by centrifugation with MEM containing 10% heat-inactivated fetal calf serum (MEM- FCS). The yield of cells
was 3-5 x 107 cells/guinea pig. According to trypan blue
exclusion , more than 95% of the cells were viable.
260
Vol . 68, N o.5
TAJUGAWA AND IMAMURA
When the cells were to be used for immunization. they
were suspended in pyrogen-free physiologic saline.
Preparation of anti-epidermal cell sera. Viable epidermal cells were injected in random-bred white female
rabbits, weighing 1.8 to 2.0 kg. Two procedures were
employed: (1) Rabbit 1 was injected intramuscularly
with 1.5 x 107 cells in 1 ml of saline to which was added
an equal volume of Freund's complete adjuvant. Two
and 4 weeks later a similar number of cells in 5 ml of
saline was given intravenously. (2) Rabbits 2 and 3
were injected intravenously with 1.5 x 107 cells in 5 ml
of saline 3 times at intervals of2 weeks. One week after
the last injection, blood was drawn from the carotid
arteries. The sera were inactivated at 56"C for 30 min
and stored frozen at -2o•c until used. The sera from
r abbits 1, 2, and 3 were referred to as AES 1, 2, and 3.
r espectively.
Pemphigus sera. The clinical and immunologic data
of 4 patients with pemphigus are described briefly in
Table III. All sera were stored at -2o•c .
Absorption of sera . An aliquot of AES was absorbed
at least 3 times with an equal volume of packed red and
spleen cells and 1 / 10 volume of thymus cells at room
temperature for 30 min. The cells were obtained from
more than 3 guinea pigs each time. AES and pemphigus
sera were eventually absorbed in the same manner
with the cells of a guinea pig used for cytotpx:icity
testing or immunofluorescence and with guinea-pig
liver powders (30 mg/ml ) which were prepared according to the method of Coons et al [141.
Cytotoxicity test. The method of Masuda et all15) was
employed. A cell suspension of 2.5 x 10"/ ml was prepared in MEM. Pooled guinea-pig sera absorbed with
guinea-pig epidermal and spleen cells were used without dilution as a source of complement (C). Four hundredths milliliter of a cell suspension, 0.05 ml of serum.
serially diluted. and 0.01 ml ofC were incubated at 37"C
for 45 min. Cell counts, living or dead , were made after
addition of0.1 ml of freshly prepared 0.2% trypan blue.
Tests always were performed in duplicate. Controls
were included in whkh the test cells were incubated
with medium instead of serum <C alone) or C (serum
alone). The results are expressd by the following formula;
primary filter and 41 and 65 secondary filter s. Photographs were taken on a Fuji or Kodak Tri-X fUm.
RESULTS
Cytotoxicity Test
Figure 1 shows the ability of absor bed AES to
lyse guinea-pig and human epidermal cells in the
presence of C as measured by trypan blue exclusion. Isolated human epidermal cells were obtained by trypsinization of the epidermis of surgically excised scar tissue from a burned patient by
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ANTISERUM DILUTION
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625
(RECIPROCAL)
Fie . 1. Complement-mediated cytotoxic activity of
rabbit anti-guinea pig epidermal cell sera <AES\ towards guinea-pig epidermal cells (broken lines\ and
human epidermal cells (solid line\. 0 , AES 1; 6., AES 2;
D, AES 3.
Percent killed cells = 100 x
1
living cells with serum + c)
TABLE
I. Titration of AES on frozen tissue sections"
- ( living cells with C alone
Fluorescein isothiocyanate fFITCJ conjugates. An antibody to rabbit IgG was prepared in a sheep and conjugated with FITC by the method of Masuda [16]. The
conjugate obtained had a F/P molar ratio of2.0 and was
used at a protein concentration of 1 mg/ml. The monospecificity was confirmed by double diffusion in agar
gel. FITC-conjugated rabbit antihuman IgG was obtained from Miles Lab. , ill., and Dakopatts, Denmark.
The commer cial conjugates were used at a dilution of
1:10.
Immunofluorescence. An indirect method was used
on frozen tissue sections as previously described 117).
For membrane staining of viable cells, 1-2 x 10" cells
were incubated with 0.2 ml of diluted sera for 30 min at
4•c, washed twice with Dulbecco's phosphate-buffered
saline containing 1% bovine serum albumin. and reacted with 0.2 ml of a conjugate for 30 min at 37•c . The
cells were washed. r esuspended in the same medium,
and wet mounts prepared. The slides were viewed with
a Zeiss fluorescence microscope equipped with a UV-5
Guinea pig
Oral mucosa
Esophagus
Epidermis:
BaM! layoc
~
Spinous layer
Granular layer
Horny layer
Hair follicle
Sebaceous gland
Thymus (Hassall's
corpuscle)
Monkey
Esophagus
Rabbit, rat, and mouse
oral mucosa
AES
AES
AES
#1
#2
#3
640
640
640
640
320
640
160
80
l60
80
160
40
40
40
40
20
40
80
80
40
20
20
20
20
20>
20>
20>
" Titer of serum at a highest dilution giving a positive reaction by indirect immunofluorescence.
May 1977
the procedure described in Materials and Methods. AES 1, 2, and 3 showed similar lytic activities
to the guinea-pig cells, while it. was impossible to
determine which type of cells, i.e. , basal, spinous,
or granular, was more susceptible to lysis by AES.
Control (C alone) always contained more than 90%
living cells. The samples that were incubated with
undiluted AES alone agglutinated to a variable
degree, while those incubated with serially diluted
AES alone resulted in less than 10% killed cells.
AES 2 showed weak cytotoxicity against human
cells. AES 1 and 3 were not tested on human cells.
It was confirmed that AES did not kill guinea-pig
spleen and thymus cells.
On the other hand. all pemphigus sera diluted
to 1:5 or more were not cytotoxic on guinea-pig or
human epidermal cells. Undiluted pemphigus sera
ANTI-GUI NEA-PIG EPIDERMAL CELL SERA
261
which were incubated with or without C agglutinated the viable guinea-pig cells to a variable degree.
I mmunofiuorescence
Frozen tissue sections. Full titrations of AES on
a variety of tissues are given in Table I. All AES
showed an intercellular staining pattern on sections of guinea-pig skin, oral mucosa, and esophagus. which extended in all layers oft..he epithelium
(Fig. 2A ). End titers in the horny layer were lower
than those in other layers of the epiderrrus. Hair
follicles and sebaceous glands also revealed intercellular staining (Fig. 2B ). No specific fluorescence was revealed in the cytoplasm of cells of the
stratified squamous epithelia or the dermal com-
FIG . 2. Indirect immunofluorescence staining of frozen sections. A: Guinea-pig oral mucosa <AES 3, 1:40. x 130).
B: Guinea-pig hair follicles and sebaceous glands (AES 2, 1:20. x 130). C: Guinea-pig oral mucosa (pemphigus
serum, case 1, 1:40, x 130). D : Guinea pig thymus <AES 2, 1:20, x 320).
262
Vol . 68, No .5
TAKIGAWA AND IMAMURA
ponents other than hair follicles and sebaceous
glands. Sections of monkey (Macaca rhesus)
esophagus reacted with AES showed an intercellular staining in all layers of the epithelium. AES
did not react to rabbit, rat, and mouse oral mucosa. When the patterns obtained with guinea-pig
oral mucosa were compared with those by pemphigus sera (Cases 1 to 4) on the same tissue, the
similarities were striking (Fig. 2C). Although absorbed AES did not react with the guinea-pig
brain, kidney. spleen, or liver, staining of the
thymus revealed whorly, homogenous fluorescent
areas corresponding to Hassall's corpuscles and
surrounding epithelial cells; no other areas of the
thymus were stained lFig. 2D ). Pemphigus sera
showed n o fluorescence in the guinea-pig thymus.
Control sera included pooled normal rabbit sera
and rabbit antifowl lgG antisera which was prepared by two injections of fowl IgG plus Freund's
complete adjuvant. Absorbed control sera gave no
reactions with any of the above tissues.
Viable cell suspensions. The guinea-pig epidermal cells treated with AES or all pemphigus sera
at 4°C and then placed with FITC-conjugates at
37°C showed fluorescence all around the cell membrane. Different staining patterns were recognized
among the various cell types. The larger cells
showed a circumferential ring pattern and the
smaller ones displayed a m·ore patchy. interrupted
ring type reaction (Figs. 3A , B). The titrations of
AES and pemphigus sera on viable cells and the
percentage of fluorescence-positive cells are given
in Tables II and ill. As the sera were diluted. the
number of positive cells was decreased. The present study could not demonstrate whether or not
fibroblasts. which might contaminate epidermal
cell preparations, reacted with AES; our immuneferritin electron microscopic study disclosed that
fibroblasts were antigen-negative.*
Effects of Further Trypsinization
Dispersed guinea-pig epidermal cells concentrated to 10u were further reacted with 1 ml of
0.03% trypsin solution at 37°C for 30 min with
occasional pipetting. After t he incubation . the
cells were washed with MEM- FCS. Viability as
counted by dye exclusion was more than 90% of
untreated cells. The viable cells were stained with
AES or pemphigus sera at a dilution of 1:10 as
described above. Although the intensity of fluorescence seemed to be unchanged, most of the cells
that reacted with AES displayed a ring staining
pattern; the interrupted pattern was rarely observed. On the other hand, fluorescent spots on the
cell surface were markedly reduced and weakened
in the cells that reacted with pemphigus sera.
DISCUSSION
Surface antigens specific for cells of the epidermis and stratified squamous epithelia were de-
* Takigawa M, Imamura S, Ofuji S: Manuscript in
preparation.
FIG . 3. Membrane immunofluorescence staining of
viable guinea-pig epidennal cells. A: Cells treated with
AES 3 (1:10, x 320l. B: Cells treated with pemphigus
serum (case 5, 1:10. x 320).
Il. Percentage of antigen-posiliue cells shou·ing
membrane immunofluorescence by AES
More than 200 cells were coun ted and the number of
fluorescent positive cells was examined.
TABLE
Dilution of serum
AES #1
AES #2
AES #3
1:160
> 90
> 90
1:320
1:640
53
50
0
> 90
33
10
0
tected by both complement-mediated cytotoxicity
and immunofluorescence using absorbed AES. Irrespective of the immunization procedures, AES 1.
2, and 3 showed similar cytolytic activity against
trypsinized guinea-pig epidermal cells. It was impossible to determine which type of cells was most
susceptible to lytic activity. Immunofluorescence
likewise did not reveal specific preference for the
different cell types even with diluted antisera. It
appears that the antigen(s) detected by AES is
present on all epidermal cells.
Immunofluorescence on frozen sections revealed
that AES did not react with other organs of guinea
pig except Hassall's corpuscles and surrounding
epithelial cells of the thymus. Thymic epithelial
cells are now considered to be of ectodermal origin
and have morphologic similarities to epidermal
cells [18]. The existence of a cross-reactive antigen
has been demonstrated in the thymic epithelial
May1977
ANTI-GUINEA-PIG EPIDERMAL CELL SERA
TABLE
Ill. I m m unoflourescence of pemphigus sera on guinea-pig epithelial tissue
Clinical diagnosis
End titer on froten oral mucosa
Percentage of antigen-positive
cells showing membrane immunofluorescence
Dilution of serum
1:10
1:20
1:40
1:80
1:160
1:320
263
Case 11'4
CIIJ!e #1
Case # 2
Case #3
P . vulgaris
P . vulgaris
P . foliaceus
P . erythematosus
160
160
320
1280
81
64
35
0
71
34
0
64
32
0
> 90
81
47
10
1:640
- = Not done .
cells and epidermal cells of the guinea pig using
antibodies to the polysaccharide of Group A streptococci I19]. Our study a lso confirmed that the
thymic epithelial cells and Hassall's corpuscle
share common antigen(s) with epidermal cells.
AES showed cross reactivity with the epidermal
cells of other species including humans and monkeys, indicating that AES was not species specific.
In huma ns, this was confirmed by the cytotoxicity
assays. In monkeys. intercellular staining was
demonstrated in all layers of the epithelium.
AES a re different from PA in that the former
are heterologous antisera and the latter are autoantibodies. A comparison between AES and PAis.
however , feasible since both are tissue specific but
not species specific as revealed by similar immunofluorescent staining patterns. One salient point
is that. unlike AES. PA did not show complementmediated cytolysis of guinea-pig and human epidermal cells despite the presence of a substance
binding to PA on guinea-pig epidermal cells as
demonstrated by membrane immunofluorescence
I20 [. It has been shown that P A do not appear
capable of complement fixation by in vitro complement staining methodology 121.221. Furthermore.
the complement system did not play a role in in
vit ro PA-mediated acantholysis wruch was induced by the incubation of the human epidermis
with pemphigus sera for more than 6 hr 123]. Thus.
PA do not show complement-mediated cytotoxicity
to guinea-pig and human epidermal cells during
short incubations.
The further treatment of isolated guinea-pig epidermal cells with 0.03% trypsin solution resulted
in a decrease in the number and intensity of PA
fluorescent spots on cell membranes. Susceptibility of antigens reactive with PA to the proteolytic
enzyme has been reported previously [24]. The
same treatme nt did not change the intensity of
fluorescence to AES although the staining pattern
was mostly the ring type.
These findings imply that the cell surface antigens reactive with AES are different from those
reactive with PA.
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