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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.
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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.
