Docsity
Docsity

Prepare for your exams
Prepare for your exams

Study with the several resources on Docsity


Earn points to download
Earn points to download

Earn points by helping other students or get them with a premium plan


Guidelines and tips
Guidelines and tips

Immunity to Hog Intrinsic Factor: Humoral and Cellular Responses to Varying Doses, Study notes of Public Health

An experiment investigating the humoral and cellular immune response to hog intrinsic factor (HIF) in rabbits using varying doses of HIF and HIF complexed to vitamin B12 as immunogens. The study found that high doses of HIF consistently produced high titres of both blocking and binding antibodies, while low doses resulted in blunted humoral responses and the presence of binding antibodies only. Cellular immunity was induced to a similar degree in both high and low dose animals. The interaction of HIF with vitamin B12 may enhance its antigenicity in vitro.

Typology: Study notes

2021/2022

Uploaded on 09/12/2022

ebby
ebby 🇺🇸

4.2

(17)

243 documents

1 / 7

Toggle sidebar

This page cannot be seen from the preview

Don't miss anything!

bg1
Inmmunology,
1973,
25,
509.
Experimental
Humoral
and
Cellular
Immunity
to
Hog
Intrinsic
Factor
P.
A.
BACON,
L.
S.
GOLDBERG
AND
R.
BLUESTONE
Departments
of
Medicine,
University
of
California
Medical
Center,
and
Wadsworth
Veterans
Hospital,
Los
Angeles,
California,
U.S.A.
(Received
30th
December
1972;
acceptedfor
publication
16
March
1973)
Summary.
Humoral
and
cellular
immunity
to
hog
intrinsic
factor
(HIF)
was
studied
in
rabbits
immunized
with
varying
doses
of
HIF
or
HIF
complexed
to
vitamin
B12.
Animals
immunized
with
large
doses
of
HIF
(1
mg)
consistently
produced
high
titres
of
blocking
and
binding
antibodies
to
IF.
At
low
dose
immunization
(10
ug),
the
humoral
response
was
obviously
blunted,
with
animals
forming
significantly
reduced
titres
of
antibodies
to
IF;
several
rabbits
in
this
group
made
only
binding
antibodies
and
one
rabbit
produced
neither
blocking
nor
binding
antibodies.
No
difference
in
humoral
responsiveness
was
noted
between
those
animals
who
received
HIF
or
an
equivalent
amount
of
HIF
complexed
to
vitamin
B
1
2
By
contrast,
cellular
immunity
as
measured
by
inhibition
of
leucocyte
migration
was
readily
induced
to
a
similar
degree
in
both
high
and
low
dose
animals,
including
the
rabbit
which
had
no
detectable
antibodies
to
IF.
When
an
intermediate
dose
of
HIF
(50
pg)
was
used
as
the
immunogen,
four
rabbits
gave
positive
leucocyte
migration
tests
only
with
HIF-vitamin
B12
complex,
suggesting
that
the
interaction
of
HIF
with
vitamin
B12
may
enhance
its
antigenicity
in
vitro.
These
data
demonstrate
that
cellular
and
humoral
immunity
to
HIF
can
be
induced
and
partially
dissociated
from
each
other
by
varying
the
dose
of
immuno-
gen.
Although
the
mechanism
responsible
for
this
dissociation
is
not
clear,
one
explanation
would
be
differing
sensitivities
of
lymphocyte-populations
to
different
doses
of
antigen.
INTRODUCTION
Autoimmune
aberrations,
including
antibodies
to
intrinsic
factor
(IF)
and
to
gastric
parietal
cells,
are
present
in
the
majority
of
patients
with
pernicious
anaemia
(PA)
(Taylor,
Roitt,
Doniach,
Couchman
and
Shapland,
1962;
Ardeman
and
Chanarin,
1963).
Moreover,
certain
patients
have
antibodies
to
IF
present
in
their
gastric
juices
(Fisher,
Rees
and
Taylor,
1966;
Rose
and
Chanarin,
1969),
and
such
antibodies
can
interfere
with
the
IF
mediated
absorption
of
vitamin
B12
(Schade,
Feick,
Muckerheide
and
Schilling,
1966).
These
findings
have
suggested
that
immune
mechanisms
may
play
a
role
in
the
pathogenesis
of
this
disorder.
Other
observations,
however,
have
indicated
that
humoral
immune
mechanisms,
i.e.
autoantibodies,
are
unlikely
to
have
significant
roles
in
either
the
genesis
or
perpetuation
of
PA;
these
include
(a)
the
observation
that
a
Correspondence:
Dr
L.
S.
Goldberg,
Department
of
Medicine,
UCLA
Medical
Center,
Los
Angeles,
California
90024,
U.S.A.
509
pf3
pf4
pf5

Partial preview of the text

Download Immunity to Hog Intrinsic Factor: Humoral and Cellular Responses to Varying Doses and more Study notes Public Health in PDF only on Docsity!

Inmmunology, 1973, 25, 509.

Experimental Humoral and Cellular Immunity

to Hog Intrinsic^ Factor

P. A.^ BACON, L. S.^ GOLDBERG^ AND^ R.^ BLUESTONE

Departments of^ Medicine,^ University^ of California^ Medical^ Center,^ and^ Wadsworth^ Veterans

Hospital, Los Angeles, (^) California, U.S.A.

(Received 30th December 1972; acceptedfor publication 16 March^ 1973)

Summary. Humoral^ and cellular^ immunity^ to^ hog^ intrinsic factor^ (HIF)^ was studied in rabbits immunized with varying doses of HIF or HIF complexed

to vitamin B12. Animals immunized with large doses of HIF (1 mg) consistently

produced high titres of blocking and^ binding antibodies^ to^ IF.^ At low^ dose

immunization (10 ug), the^ humoral^ response was^ obviously blunted,^ with^ animals

forming significantly reduced^ titres of antibodies^ to^ IF; several rabbits in this^ group

made only binding^ antibodies^ and^ one^ rabbit^ produced^ neither^ blocking^ nor

binding antibodies. No difference in humoral responsiveness was noted between

those animals who received HIF or an equivalent amount of HIF^ complexed to

vitamin B 1 2 By contrast, cellular immunity as measured by inhibition of leucocyte migration was readily induced to a^ similar^ degree in^ both^ high and low dose animals, including the^ rabbit which^ had^ no^ detectable antibodies^ to^ IF.^ When an (^) intermediate dose of HIF (50 (^) pg) was used as the immunogen, four rabbits gave

positive leucocyte migration tests^ only^ with^ HIF-vitamin^ B12 complex,^ suggesting

that the interaction of HIF with vitamin B12 may enhance its antigenicity in

vitro. These data demonstrate that cellular and humoral immunity to HIF can be induced and partially dissociated from each other by varying the dose of immuno- gen. Although the mechanism responsible for this dissociation is^ not^ clear,^ one explanation would be differing sensitivities^ of^ lymphocyte-populations to^ different doses of (^) antigen.

INTRODUCTION

Autoimmune aberrations, including antibodies to intrinsic factor (IF) and to gastric parietal cells, are present in the majority of patients with pernicious anaemia (PA) (Taylor, Roitt, Doniach, Couchman and Shapland, 1962; Ardeman and Chanarin, 1963). Moreover, certain patients have antibodies to IF present in their^ gastric^ juices (Fisher, Rees and Taylor, 1966; Rose and Chanarin, 1969), and such antibodies can interfere with the IF mediated absorption of vitamin B12 (Schade, Feick, Muckerheide and Schilling, 1966). These findings have suggested that immune mechanisms may play a (^) role in the pathogenesis of this disorder. Other observations, however, have indicated that humoral immune mechanisms, i.e. autoantibodies, are unlikely to have significant roles in either the genesis or perpetuation^ of^ PA;^ these^ include^ (a)^ the observation^ that a Correspondence: Dr L. S. Goldberg, Department of Medicine, UCLA Medical Center, Los Angeles, California 90024, U.S.A.

509

P. A. Bacon, L. S. Goldberg and R. Bluestone distinct (^) population of patients with PA (^) has neither antibodies to IF (^) nor to (^) parietal cells and (^) (b) the (^) finding of PA (^) in (^) patients with (^) acquired agammaglobulinaemia (^) who (^) are unable to elicit a humoral (^) antibody (^) response. Thus, if immunological (^) phenomena (^) are

intimately involved^ in^ this disorder, it^ may be^ the^ cell-mediated immune^ mechanisms rather than humoral (^) responses which are of (^) importance. Indeed, several (^) recent (^) reports have demonstrated the (^) presence of (^) cell-mediated immunity to IF (^) in (^) patients (^) with (^) PA

(Rose, Chanarin,^ Doniach,^ Brostoff and^ Ardeman, 1970;^ Finlayson, Fauconnet and Krohn, 1972). In^ these^ reports, cellular^ immunity to^ IF^ as^ measured^ by (^) lymphocyte production of^ migration inhibitory factor^ did^ not^ precisely correlate^ with^ the (^) presence

of serum antibodies to IF, that is, cellular immunity to IF may exist in the presence or

absence of^ humoral^ antibodies (^) to (^) IF. Assuming cell-mediated immunity is (^) important (^) in PA, these^ observations^ would^ offer^ an (^) explanation for^ the^ presence of^ PA^ in^ patients (^) with or without autoantibodies and in those with agammaglobulinaemia. Limited information is (^) available (^) regarding factors involved in the (^) humoral (^) and cellular immune (^) response IF. When (^) very small doses of human IF (^) used as (^) the

immunizing antigen,^ rabbits^ will^ produce^ only^ blocking^ antibody^ to IF, i.e. antibodies

directed (^) against the vitamin B1 2-combining site of IF^ (Samloff and (^) Turner, 1968). When larger doses^ of^ human^ IF complexes of^ IF-vitaminB12 are^ employed^ immunogens, rabbits (^) produce both^ blocking antibody and (^) antibody directed (^) against the (^) IF-vitamin

B12 complexes, i.e.^ binding antibody. These^ observations^ suggested that^ the dose of

IF, and^ perhaps its^ configurational change when^ attached^ to^ vitamin^ B12, determine its

antigenicity within^ the^ humoral^ system. No^ such^ data^ are^ available^ the^ cellular

immune (^) response to IF. In (^) the (^) present (^) investigation, the humoral and (^) cellular (^) immune

response to^ IF^ was^ studied^ in^ rabbits^ using^ varying^ doses^ of^ IF^ and^ IF-vitaminB

complexes as immunogens.

MATERIALS AND^ METHODS Animals, antigens and antisera

New Zealand white rabbits weighing approximately 3 kg were used. Lyophilized hog

intrinsic factor (^) (HIF-No. 3908C) was (^) kindly supplied by Dr Leon (^) Ellenbogen, Lederle Laboratories, Pearl^ River, N.Y. (^) Ninety-two per cent^ of^ the^ vitamin B1 2-combining

capacity of this preparation was blocked by the addition of either human autoantibody

to IF or rabbit antiserum to HIF, indicating that the material consisted almost entirely

of IF with minimal non-IF-vitaminB12 binders. Radio-labelled cobalt60-vitaminB

containing 0 75 pCi/0 95 pg with a specific activity of 1 26 pCi/ml was purchased from

E. R. Squibb and Son, New York, N.Y. and [59Co]B12 (Rubramin) was obtained from

Parke-Davis Co. Goat antiserum to rabbit IgG, Miles Laboratories, Kankakee, Ill.,

was shown to be monospecific in that it gave single precipitin lines when tested by

immunoelectrophoresis and agar gel double diffusion against whole normal rabbit serum

and isolated rabbit IgG.

Antigens for immunizations were prepared in the following manner. Lyophilized

HIF, 10, 50 and 1000 ug, were dissolved in 1 ml of saline and incubated with excess vitamin

[59Co]B12 to saturate all vitamin B1 2-binding sites. Unbound vitamin 12 was removed

by absorption with albumin-coated charcoal pellets. Identical solutions of HIF, 10, 50

and,g/ml, 1000 were prepared with no vitamin 1I2 added. Prior to immunization, each

HIF preparation was mixed with an equal volume of Freund's complete adjuvant.

512 P.^ A.^ Bacon,^ L.^ S.^ Goldberg^ and^ R.^ Bluestone with HIF, 1000 jug, and three with HIF, 1000 ug, complexed to^ vitamin^ B12.^ Group^ 2: Three rabbits received HIF, 50^ Mtg, and three^ received^ HIF 50^ pg,^ complexed^ to^ vitamin B1 2. Group 3:^ Three^ rabbits were^ injected^ with HIF, 10^ jg,^ and three with^ HIF,^10

yug, complexed^ to^ vitamin^ B1^ 2.^ Animals^ immunized^ with^ HIF-vitamin^ B12^ complex

received 1000 Mg of vitamin [59Co]B12 intramuscularly prior to immunization. Four

rabbits which were immunized with Freund's complete adjuvant alone served^ as^ controls. Animals were bled before immunization and at 2, 4 and 6 weeks after^ immunization. The serum specimens were tested for blocking and^ binding^ antibodies^ to IF.^ Leucocytes were separated from each blood specimen and used in the^ leucocyte migration^ test (vide supra). After 6 weeks, cellular immunity^ was also^ assessed^ by^ the^ intradermal^ injec- tion of HIF, 50 Mtg and HIF, 50 (^) yg, complexed^ to^ vitamin B12.^ At 24^ and^48 hours,^ the area of erythema and^ induration^ was^ measured.^ An^ indurated^ area of^5 mm or^ greater was considered a^ positive^ test.

RESULTS

RABBITS IMMUNIZED WITH HOG IF OR HIF-B12 1 MG Group 1 These animals produced^ high titres of^ both blocking^ and^ binding^ antibodies^ to HIF (Table 1).^ Blocking^ activity^ ranged from^ 21 to 27^ ng/0^ 1 ml^ with^ the^ greatest^ titres occurring 4 weeks^ post-immunization.^ Binding antibodies^ were^ also^ detected^ in^ titres

of 1:64-1:1024.

Cellular immunity to HIF was also present in these rabbits. Significant inhibition^ of leucocyte migration was observed at 4 and 6 weeks^ post-immunization^ when either

TABLE 1 HUMORAL AND CELLULAR IMMUNITY TO HOG^ INTRINSIC^ FACTOR

Migration index using Blocking Binding different antigens Animal Immunogen antibody* antibodyt HIF HIF-B Rabbit No. 1 HIF,^ 1 mg 26 256 0-72^ 0- 2 HIF, 1 mg 21 64 0-73^ 0- 3 HIF, 1 mg 25 1024 0-71^ 0- 4 HIF, 1 mg-B12 27 1024 0-71^ 0- 5 HIF, 1 mg-B12 24 256 0-63^ 0- (^6) HIF, 1 mg-B12 25 256 0-7 0- Rabbit No. 7 HIF, (^50) ,g 10 16 1-04 0- 8 HIF, 50,pg 24 64 0-91^ 0- 9 HIF, 50 (^) ,pg 19 64 0-76^ 0- 10 HIF, 50 pg-B12 16 64 0-98^ 0- 11 HIF, 50 pg-B12 26 256 0-97^ 0- 12 HIF,^50 ,pg-B,2 19 64 0-8^ 0- Rabbit No.^13 HIF,^10 ,pg 6 16 0-61^ 0 57 14 HIF, (^10) jug 0 4 0-54^ 0- 15 HIF, 10 pig 0 0 0-76 0- 16 HIF, 10 ug-B12 9 16 0-51^ 0- 17 HIF, 10 Ug-B12 3 4 0-55 063 18 HIF, 10 pg-B12 0 4 0-75 0-

  • (^) Expressed as (^) ng of vitamin (^) B12 uptake blocked by 0- 1 ml serum. t Expressed as^ reciprocal of titre.

Humoral and Cellular Immunity to HIF

HIF or HIF complexed to vitamin B12 was used as the antigen. Migration indices (MI)

4 weeks after immunization were 0-63-0-73 with HIF and 0-7-082 with HIF-vitamin B12 (Table 1). Similar values were noted at 6 weeks. Animals immunized with either HIF or HIF-B12 complex gave positive skin tests when these antigens were administered intradermally. The areas ofinduration at 48 hours ranged from 8 to 18 mm. No significant differences in^ humoral and^ cellular immune^ responses were^ observed between those

animals injected with HIF and those with HIF-vitamin B1 2*

RABBITS IMMUNIZED WITH IF OR HOG-B12, 50 UG Group 2 Significant titres of^ blocking and^ binding antibodies^ to^ IF^ occurred^ in^ this^ group.

Blocking activity in these sera ranged from 10-26 ng/0 1 ml with binding antibody titres

of 1: 16-1 :256 (Table 1). The highest antibody titres again were detected 4 weeks after

immunization. Cellular immunity to IF was also present in these six animals. Interestingly,

four ofthe six rabbits (two immunized with HIF, two with^ HIF-B12) gave positive leucocyte

migration tests only where^ HIF-vitamin^ B12 was^ used^ as^ the^ antigen in vitro^ (MI of 07-

0.8). Of^ the^ remaining two^ rabbits, No.^9 showed^ significant inhibition^ with^ both HIF

and HIF-B12 (MI of 0-76 and 0.8), while rabbit No. 12 gave a positive test only when

tested with HIF (MI-0.8). Skin tests were positive with indurated areas of greater than

5 mm in all rabbits except No. 7.

RABBITS IMMUNIZED WITH HIF OR (^) HIF-B12, 10 IG

Group 3 Five of the six rabbits in this group formed either blocking and/or binding antibodies to IF. The sole exception, rabbit No. 15 which was immunized with 10 ug HIF, produced neither blocking or binding antibodies. Two animals, No. 14 and 18, formed binding but not (^) blocking antibodies. The titres of (^) blocking and (^) binding antibodies were signific- antly less than those observed in rabbits immunized with larger doses of HIF. By contrast, several animals gave positive tests in the leucocyte migration assays at

2 weeks after immunization and all were strikingly positive at 4 weeks. The migration

indices at 4 weeks post-immunization ranged from 0-54-0-76 when HIF was used as the antigen and from 0-54-0-68 with HIF-vitamin B12. Animal No. 15 which did not form

antibodies to IF showed MI of 0-76 and 0-65 when tested against HIF and HIF-B

respectively. Skin tests were also positive in these animals with areas of induration of 5- mm observed at 48 hours. Rabbits injected with Freund's adjuvant alone failed to produce antibodies to IF. These animals did not demonstrate cellular immunity to HIF when tested in the leucocyte migration test or by intradermal administration to HIF.

DISCUSSION

The results of these experiments demonstrate that humoral and cellular immunity to IF can be induced in rabbits. Humoral immunity to IF appeared to depend in part on the dose of immunogen. When rabbits were immunized with large amounts of HIF (1 mg), high titres of blocking and binding antibodies were consistently produced. At low

Humoral and Cellular Immunity to HIF 515

and humoral immunity has been offered as an explanation for the paradox of autoimmune disease occurring in agammaglobulinaemic subjects. A similar phenomena would^ also explain the presence of autoimmune disease in the absence of autoantibodies. Experi- mental dissociation of humoral and cellular responses to^ various^ antigens^ such^ as^ IF^ would lend support to these concepts.

ACKNOWLEDGMENTS

Jo Ellen Cunningham, Gail Davis, Miss S. Brady and Mr W. Mills provided skilled technical assistance. This work was supported by grants from the^ United States Public Health Service (AM CA 15220-01 and^ GM^ 15759)^ and^ from^ the^ Arthritis^ and^ Rheu- matism Council of Great Britain.

REFERENCES ARDEMAN, S. and CIHANARIN, I.^ (1963). 'A method^ for the assay of human gastric intrinsic factor and for detection and titration of antibodies against intrinsic factor.' Lancet, ii, 1350. FINLAYSON, N. D. C., FAUCONNET, M. H.^ and KROHN, (1972). 'In vitro^ demonstration of^ delayed hyper- sensitivity to gastric antigens in pernicious anemia.' Amer. (^) J. digest. Dis., 17, 631. FISHER, J. M., REES, C. and TAYLOR, K. B. (1966). 'Intrinsic-factor antibodies in gastric juice of pernicious anaemia patients.' Lancet, ii, 88. GOLDBERG, L. S.,^ SAMLOFF, I. M. and^ BARNETT, E.^ V. (1969). 'Studies on the antigenic structure of human and hog intrinsic factors.' Clin. exp. Immunol., 5, 371. GOTTLIEB, C., LAU, K.-S., WASSERMAN, L. R. and HERBERT, V. (1965). 'Rapid charcoal assay for intrinsic factor (IF), gastric juice unsaturated B binding capacity, antibody to IF, and serum unsaturated B12 binding capacity.' Blood, 25, 875. PLAYFAIR,J. H. L. and PURVEs, E. C. (1971). 'Antibody formation by bone marrow cells in irradiated mice. I. Thymus-dependent and thymus-independent responses to sheep erythrocytes.' Immunology, 21, 113.

ROSE, M. S. and CHANARIN, I. (^) (1969). 'Dissociation of intrinsic factor from its antibody: Application to study of pernicious anaemia gastric juice specimens.' Brit. med. (^) J., 1, 468. ROSE, M. S., CHANARIN, I., DONIACH, D., BROSTOFF, J. and ARDEMAN, S. (1970). 'Intrinsic factor anti- bodies in absence ofpernicious anaemia.' Lancet, ii, 9. SAMLOFF, I. M. and BARNErr, E. V. (1965). 'Identi- fication of intrinsic factor (^) autoantibody and intrinsic factor in man by radioimmunodiffusion and radioimmunoelectrophoresis.' (^) J. Immunol., 98, 558. SAMLOFF, I. M. and TURNER, M. D. (1968). 'Rabbit blocking and (^) binding antibodies to human intrinsic factor and (^) intrinsic (^) factor-B112 complex.'J. Immunol., 101, 578. SCHADE, S. G., FEICK, P., MUCKERHEIDE, M. and SCHILLING, R. F. (1966). 'Occurrence in gastric juice of (^) antibody to (^) a complex of intrinsic factor and vitamin (^) B52.' New Engl. J. Med., 275, 528. TAYLOR, K. B., Rorrr, I. M., DONIACH, D., COUCHMAN, K. G. and SHAPLAND, C. (^) (1962). 'Autoimmune phenomena in pernicious anaemia. Gastric anti- bodies.' Brit. med. (^) J., 2, 1347.