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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.
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Inmmunology, 1973, 25, 509.
P. A.^ BACON, L. S.^ GOLDBERG^ AND^ R.^ BLUESTONE
Hospital, Los Angeles, (^) California, U.S.A.
Summary. Humoral^ and cellular^ immunity^ to^ hog^ intrinsic factor^ (HIF)^ was studied in rabbits immunized with varying doses of HIF or HIF complexed
produced high titres of blocking and^ binding antibodies^ to^ IF.^ At low^ dose
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
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.
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
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
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
IF, and^ perhaps its^ configurational change when^ attached^ to^ vitamin^ B12, determine its
immune (^) response to IF. In (^) the (^) present (^) investigation, the humoral and (^) cellular (^) immune
complexes as immunogens.
MATERIALS AND^ METHODS Animals, antigens and antisera
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
and isolated rabbit IgG.
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
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.
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
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-
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
RABBITS IMMUNIZED WITH IF OR HOG-B12, 50 UG Group 2 Significant titres of^ blocking and^ binding antibodies^ to^ IF^ occurred^ in^ this^ group.
immunization. Cellular immunity to IF was also present in these six animals. Interestingly,
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
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
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.
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.
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.