Effect of Prolonged Administration of Homologous and Heterologous Intrinsic Factor Antibodies on the Parietal and Peptic Cell Masses and the Secretory Function of the Rat Gastric Mucosa

Vol. 69, No.2

GASTROENTEROLOGY69: 396-408, 1975

Printed in U.S.A.

Copyright© 1975 by The Williams & Wilkins Co.

EFFECT OF PROLONGED ADMINISTRATION OF HOMOLOGOUS AND HETEROLOGOUS INTRINSIC FACTOR ANTIBODIES ON THE PARIETAL AND PEPTIC CELL MASSES AND THE SECRETORY FUNCTION OF THE RAT GASTRIC MUCOSA MASAMI INADA, M .D., AND GEORGE B. JERZY GLASS, M.D.

Gastroenterology Research Laboratory, Department of Medicine, New York Medical College, New York, New York

In order to determine the possible effects of the circulating intrinsic factor antibodies (IF A) on gastric morphology and secretory function, four groups of 12 rats each received intravenous injection daily for 8 to 12 weeks of immunoglobulin G (lgG) fractions separated on DEAEcellulose columns from various sources: (1) sera of patients with pernicious anemia, containing both IF:A and parietal cell antibodies (PCA), (2) sera from patients with atrophic gastritis, containing parietal cell antibody only, and (3) and (4) sera of rabbits immunized with semi purified human or rat intrinsic factor (IF). In addition three control groups of 12 rats each received intravenous injections daily for 8 to 12 weeks of either (5) saline or (6) and (7) lgG processed from human or rabbit normal sera. Still another group of 12 rats (8) did not receive any injections whatsoever for the same duration of time. One-third of the rats were intubated biweekly after histamine stimulation and the hourly outputs of HCl pepsin, and IF were determined. At conclusion of the experiments, rats were killed, the mucosal surface and thickness of the mucosa were measured, and parietal cell and peptic cell masses were counted. The control groups showed either progressive growth of the cellular mass in gastric mucosa and increase of the HCl, pepsin, and IF outputs, or no significant changes. In contrast, rats injected with lgG containing IFA to human or rat IF showed a statistically significant thinning of the gastric mucosa, reduction of pept'ic_cells, which are known to secrete IF in this species, and corresponding decrease in the outputs of pepsin and IF. These became reduced by about 50% from initial values, and by 62 or 75%, respectively, when compared to rats injected with normal human or rabbit lgG's. In rats injected with IgG's from pernicious anemia sera, which contained both IFA and PCA, the outputs of IF, pepsin, and HCl decreased significantly, as well as the peptic and parietal cell masses. The rats injected with PCA only demonstrated thinning of the gastric mucosa, reduction of parietal cell mass, and a significant decrease of the HCI output. These findings imply an active role of the circulating gastric Received September 27, 1974. Accepted March 5, 1975. Address requests for reprints to: Dr. George B. Jerzy Glass Gastroenterology Research Laboratory, Department of Medicine, New York Medical College, New York, New York 10029. Supported by Research Grant AM-00068-19 to 21 from the Nationa l Institute of Arthritis, Metabolism 396

and Digestive Diseases, National In stitutes of Health, United Sta tes Public Health Service . Dr. Inada is a Former Research Fellow in the Gastroenterology Research Labora tory, Department of Medicine, New York Medical College, New York, New York. His present address is: Department of Geriatrics, Kyoto University Hospital, Kyoto, Japan.

August 1975

EFFECT OF !FA ON GASTRIC FUNCTWN

:397

autoantibodies in the reduction of the peptic and parietal cells masses and gastric secretory failure. Although animal experiments are not directly applicable to humans, the possible implications of this work for the natural history of gastric atrophy in man are self-evident. The role of circulating intrinsic factor antibodies (IFA) in the development of atrophic lesions of the gastric mucosa and natural history of pernicious anemia (PA) has been controversial. One group of researchers maintains that circulating IF A are the consequence of injury to the gastric mucosa that result in release of intrinsic factor (IF) ." into the circulation. 1 • 3 Since there is no immunological tolerance to IF, its passage into circulation would result in formation of an antibody whose role in influencing the course of the disease is here considered to be insignificant. According to other investigators,<- 7 IFA may actively participate . in the development and maintenance of PA, yet this participation would be directed exclusively toward the maintenance of vitamin B 12 deficiency . This would be caused by passing of IF A Into _t_b._g_ __ g~strointestinal tract and neutralizingJF in the gastric or intestinal lnm,e_n.. ~~~Another mechanism of active participation of IFA in the development of PA has been suggested by the demonstration that an antigen-antibody reaction may occur in vitro in the parietal cell of man between the IF and IF A. 1 2 • 1 3 As shown in our laboratory by immunofluorescent technique, thi~om~x binds complement which [email protected]?Ults in formation of an antigenantibo
cells and of their secretory output. Preliminary reports on this work have been published in abstract form 15 - 17 and referred to in some of the recent publications of ours. 18 · 10

Materials and Methods Outlay of the study. Ninety-six female Sprague-Dawley rats, from the same breed and weighing 260 to :120 g, were divided into eight groups of 12 rats eac h. or these, four ~-:roups served as controls a nd received: ( l) no injections whatsoever or daily intravenous injections of (2) 0.5 ml of' normal saline, (:!) 4 mg of' norn\al human immuno~-:lobulin G (lgG) in
oc

398

INADA AND GLASS

Ill), and the Sorensen glycine buffer. Three times crystallized bovine pepsin (General Biochemicals, Chagrin Falls, Ohio) was used as a standard. Intrinsic factor in rat gastric juice was measured using the zirconyl chloride gel method at pH 5.0 as described by Hansen et al. 23 The IF concentrations were expressed in units, i.e., nanograms of B 12 bound by 1 ml of gastric juice. The Z-gel was obtained from Matheson, Coleman and Bell, Division of the Matheson Co., Inc., East Rutherford, New Jersey. 57 Co-labeled B 12 , having the specific activity of 20 f.1C per /Jg, was purchased from Amersham-Searle Radiochemicals (Amersham, England) . The measurements of radioactivity were done in the automatic well counter with an automatic recording device and printer. Measurements of the mucosal thickness and surface. Upon conclusion of the experimental period of 8 or 12 weeks, thE rats were fasted overnight and killed by inhalation of ether in hermetically closed glass containers . Exsanguination was then performed immediately from the left ventricle, and the stomachs were removed and cut open along the greater curvature. The mucosal surfaces were immediately washed with normal saline buffered to pH 7.1, and the stomachs were stretched and pinned on cardboard with the mucosal surface facing upward. The outer periphery of the glandular stomach was traced onto the cardboard and the mucosal area was measured on the tracing with a planimeter. Stretched and pinned to the cardboards, the stomachs were fixed in 4% formalin at 4°C for 24 hr. The outlines of the mucosal surface, which had shrunk in the formalin, were again traced on cardboard, and their surfaces remeasured with the planimeter. From the two surface values the shrinkage coefficients were calculated. Six 3-mm wide horizontal strips were cut from each fundal area (three strips on each side) measuring from lesser to greater curvature with the distances of 3 mm be tween upper and middle and middle and lower strips. The strips were de hydra ted with a I coho I and chloroform and embedded in paraffin. From each of the six strips, sever a 1 5-f.l thick sections were cut perpendicularly to the mucosal surface along the entire length of each of the strips. These were stained alternatively with hematoxylin and eosin, 0.1% toluidine blue (National Aniline Co.) buffered to pH 3.5 with citrate-phosphate buffer, and Zimmerman 's aurantia stain as modified by Marks and Drysdale. 24 Mucosal thickness was microscopically measured using 100 x magnification and a grid

Vol. 69, No.2

(0.7 by 0.7 mm) calibrated into 0.1-mm spacings. Measurements were made at three points in each of six strips; ( 1) points A, located laterally from the medial ends of the strips at a distance equal to one-tenth of the total length of the strip; (2) points B, located exactly at the middle point of each strip; (3) points C, located medially from the lateral ends of the strips at a distance of one-tenth of the total length of the strip. The mean mucosal thickness of the fixed gastric mucosa was then calculated from these 18 measurements. Using the shrinkage coefficient, this value was converted into the mucosal thickness of the nonfixed stomach. Measurements of the parietal and peptic cell masses. Parietal cell mass was measured by the method of Bralow et al, 2 5 as slightly modified in our laboratory? 0 The peptic cell mass was determined according to Crean 26 as adapted to our study. Cellular measurements were taken at the same points at which mucosal thickness was measured. Counting of peptic cells was done only on the cells with toluidine blue dark stained nuclei, starting at the point just below the level of the mucous neck cells. This was done to avoid inclusion of the mucous neck cells in peptic cell counts and resulted in a somewhat lower peptic cell count, as compared to that of parietal cells, than usual. Peptic cells were counted after deparaffinizing, on toluidine blue-stained preparations (30-min stain, 2 x 2 dips in 95% alcohol, clearing in two changes of xylene, 2 and 5 min each and mounting in Permacell). Parietal cells were counted on aurantia-periodic acid-Schiff-stained sections and only the yellow or orange stained cells having the characteristics of parietal cell cytoplasm and a large round nucleus were included into the counts. The cells were counted with the use of a grid mounted to the eyepiece which formed a square 0.29 by 0.29 mm at 250 x magnification . All parietal and peptic cells were counted in a 0.29-mm wide column by moving the grid from the area where the crypts ended down to the bottom of gastric glands, i.e., through the entire depth of gastric glands. Eighteen such counts were taken from each of the gastric mucosal areas in which the mucosal thickness was measured. The total cell counts both of parietal and peptic cells were calculated according to the following equation:

TCC ;

LI S cc X MA X cs =-----18 X 0.29 X 0.005

where TCC is the total cell count per rat stomach, L: 1 s CC is the total cell count of the 18

August 1975

EFFECT OF !FA ON GASTRIC FUNCTION

399

areas of the glandular rat mucosa counted, MA rabbits developed a n adequately high titer of is the total mucosal surface in square milliliters IF A's, 40 ml of blood were drawn from each and before shrinkage of the glandular portion of the the sera were stored at - :20°C. The immunized rat stomach, and CS is the coefficient of rabbits produced IFA of both the bind in~-: and shrinkage, with 0.29 the width of the grid in blocking types, at the titers of 60 to 100 ng U millimeters and 0.005 the thickness of the per ml and 20 ng U per ml, respectively. All section in mm . these sera were PCA-negative. The mucosal thickness did not enter into the Human IF-B, 2 complex used for immunizacalculation, since the cells were counted tion of rabbits was prepared, using the method through the entire depth of the gastric glands in of Yamaguchi et al. 32 (fig. l) from a pool of the 18 areas counted. acid human gastric juices collected for :10 min Normal human sera were obtained from the after subcutaneous injection of 50 m~-: of Hisblood bank, tested for PCA and IF A, and found talog. The pH of the juices was first raised to negative for these antibodies (for methods see 10.0 with 2 N NaOH solution for lfi min to below). destroy peptic activity and then brought top H Sera positive for PCA and ne~ative for !FA 7.0 and stored at - 20°C. were obtained from 42 patients with atrophic Anti-rat !FA containin~ rabbit sera in the gastritis and other gastrointestinal diseases. total volume of 1200 ml were obtained from They were made into several pools and frozen at rabbits immunized with rat IF preparations. -20° until used for preparation of IgG. Im- The immunization schedule was similar to that mediately after thawing, each of the pools used for immunization of rabbits with human was tested again for PCA on hum an and rat IF-B, 2 complex . All the sera were PCA-negagastric mucosa and the poo Is repeatedly tive, and had the titers of the binding type !FA positive were used in the preparation of of 20 to 56 ng U per mi. PCA-posi tive IgG. Sera positive for !FA and R at IF-B, 2 complex used for immunization of PCA were obtained from P A patients diagnosed rabbits was obtained from rat gastric juices by hematologica l and isotopic methods. These collected for 30 min after histamine injection included vitamin B 12 absorption tests, using the (0.1 mg per kg of weight) . The individual double label hepatic uptake method of neutralized gastric JUICes were rapidly Weisberg and Glass, 27 and IF assays in the centrifuged in a refrigerated centrifuge and the gastric juice of these patients by the Yamaguchi supernatants were pooled and stored at -- 20°C. and Glass 28 modification of the Ardeman and Before use the pool was analyzed by the Z-ge l Chanarin charcoal method 2!> or by the method 23 for its IF content, saturated with K,"· zirconium gel technique of Hansen et al 23 The twice the amou nt of its B," binding capacity. sera were stored at - 20°C until processed. and concentrated 10-fo ld at 4°C a~-:ains t All rabbit sera (including normal ones) were Carbowax 10,000. The excess B,, was reobtained from several healthy and untreated moved by gel filtration on Sephadex G-2fi colNew Zealand rabbits weighing 2 to 3 kg. Normal umn (2.5 by 20 em). Stepwise elution on Binsera were negative for PCA and IFA. Rex column was then performed according to Antihuman IFA- containin~ rabbit sera were the technique described in our laboratory." The obtained from rabbits immunized with human second radioactive peak was collected, IF-B, 2 complex, containing 52 U of IF in 1 ml of concentrated to 20-ml volume on Carbowax saline, saturated with 57 CoB, 2 , and mixed 10,000, thoroughly dialyzed against severa l with an equal amount of complete Freund's changes of distilled water, and stored in 2-m l adjuvant. The sera were injected at weekly aliquots at - 20° until used. When normal rat gastric mucosae were used intervals of 4 weeks, first in the footpads and later subcutaneously and intramuscularly into as a source of IF for the rabbit immunizations other parts of the rabbits' body. If needed, the fundic mucosae were stripped from the additional injections of IF-B , 2 complex were glandular portions of 20 rat stomachs, homogegiven at weekly intervals. Prior to immuniza- nized , and then suspended and extracted into tion, the B, 2 binding capacity of the rabbit 2% NaCl solution at 4°C. Its IF content was aswas saturated by intramuscular injection of sayed by the Z-gel method at pH 5.0. The ma100 J.Lg of nonradioactive cyanocobalamin. At terial was saturated with radioactive B 12 and 2 and 4 weeks after the last immunization, the excess B 12 was removed on Sephadex G-2fi. blood was drawn from the rabbits and the titers This was followed by elution on Bio-Rex column of blocking and binding IFA against huma n IF and collection of IF fraction, as just described. Assay of PCA in human and rabbit normal were determined by the Z-gel techniques of Hansen et ai.'" and Jacob et al" · After the and immune sera was done on normal human

INADA AND GLASS

400

Vol. 69, No.2

(B) Se hadex G-25 Column 3. concentrate outer volume by evaporation

4. equilibrium dialysis against Na citrate

(0.2~,pH

3.0)

5.4) effluent by evaporation (D) Sephadex G-100 Column 6. concentrate SlcoD12-IF peak

FIG. 1. Fractionation of intrinsic factor (IF) from human gastric juice.

and /or rat gastric mucosae by Coons' indirect immunofluorescence "sandwich technique" as applied to gastric mu cosa by Taylor et aP 3 and Irvine. 34 Fluoresce inated a nti-human and anti-rabbit IgG globulins (Hyland Division of Travenol Laboratories, Los Angeles, Calif.) had a ratio of 5 to 7 mg of Fluorochrom to 1 g of protein. Four-micron thick tissue sections of human or rat gastric mucosa were cut in a cryostat at - 20°C and transferred to air-dried s !ides to be placed in a moist chamber. The immunofluorescence was read in the lightfluorescent microscope with the use of a D 0.080 condenser. Color pictures were taken using Anscochrome 2-200 day light color film , 135 g.a.f. Assay of !FA of the binding type was done by the zirconium gel method of Jacob et a!. 31 at pH 6.25 and retested by the immunodiffusion method. This was followed by radioautography and testing for peripheral immunofluorescence of parietal cells in the indirect Coons' test, as described before. 13

Processing of lgG from normal and immune human and rabbit sera . This was d one using the method of Vaerman et al. 35 as modified in our laboratory .""· 3 6 Six groups of sera mentioned above were precipitated by saturation with ammonium sulfate at 4°C for 2 hr. The precipitate was separated by centrifugation at 4°C and 10,000 rpm for 10 min, redissolved in normal saline, dialyzed at 4°C against normal saline for 24 hr, and against 0.01 M phosphate buffer (pH 7.5) for the subsequent 24 hr with frequent saline and buffer changes, and centrifuged again at 10,000 rpm at 4 °C for 10 min . Fifty to 70 ml of supernatant, derived from 150 to 170 ml of serum, were applied to the top of the 4.5 by 30 em glass column packed with a recycled diethylaminoethylcellulose (Serva) that was washed prior to use with 0.01 M phosphate buffer, pH 7.5. The column was eluted at soc with the same buffer at a flow

rate of 30 to 40 ml per hr. The effluents were collected into 10-ml fractions by means of a linear fraction collector provided with an automatic volume counting unit. The optical density of each fraction was read in a DB Beckman spectrophotometer at 280 nm and traced on paper. The effluent fractions corresponding to the first sharp peak of optical density that contain lgG fraction 35 • 36 were pooled, dialyzed against distilled water at 4°C for 24 hr, and lyophilized . The material trailing behind was discarded. The purity of the IgG fractions obtained was tested by immunoelectrophoresis on agar gel, in verona! acetate buffer (pH 8.6) , against antisera to human and rabbit sera, using Scheidegger 's method " and thiazine red stain. As shown before 20 the material in the first peak gave only one line of lgG on immunoelectrophoresis.

Results Effect of lgG Ln}ections on the weight. Rats not injected or injected with saline showed an increase in weight by 10 to 20%. Probably due to the runting effect of foreign protein injections , rats receivin g lgG from various sources showed less increase in weight ( ± 6%) which did not differ significantly in various groups. The effects of IgG injections on gastric mucosal thickness are shown in figure 2. The~rlJZ:.9J.g~Q.!:U?_Qf rats no_!j.!_lj_§_<2ted with lgQ._b~Q.._§imilar thickness as controls injected with saline or normal human or rabbit lgG's. Rats \!!j~cted with PCA- and human_J.Ef..-containing human lgG, or anti-rat_IFA-containing rabbit lgG , showed a 25 to 35% decrease, however, m mucosal thicknessJ_®~.!!_!QJ:~~ range of 300 to 330 J.L. The difference was statistically highly

August 1975

EFFECT OF !FA ON GASTRIC FUNCTION

significant with P values below 0.05 and 0.01, respectively. The reduction in mucosal thickness in animals injected for 8 weeks with antibodies-containing IgG's were less marked than in the .12-week injected group, and amounted to 15% as compared to controls. Controls

)J

500 400 300 200 100 0

No

Normal

Sol1ne

PCA

Injections

PCAond Anlt·humon

Normal Anlt· human

IF

IF

Anhrot

IF

FIG. 2. Gastric mucosal thi c kness in control rats and those injected daily for 12 weeks with IgG 's of various sources (means of 8 rats with standard error). Difference between controls and rats injected with parietal cell antibodies (PCA) and anti-human intrinsic factor (IF) antibodies or anti-rat intrinsic factor (IF) rabbit antibodies was highly significant (P < 0.05 and < 0.01 , respectively) .

When mean mucosal thickness was calculated per 1 kg of weight, the differences between animals injected with antibodies and controls still amounted to 15 to 20
Human lgG

150

401

Ig G

~Parietal cell mass

c§ Peptic cell moss (/)

z

0

:::i

100

...J

~ ~ (/)

...J ...J

w u

50

0

Normal

PCA

PCA and Anli·humon

Normal

Antihuman

Antirot ·.

IF IF lF FIG. 3. Parietal cell and peptic cell masses in rats injected daily for 12 weeks with lgG 's of various sources

(means of 8 rats with standard error). Difference between control rats injected with normal human lgG and those injected with parietal cell antibodies (PCA)-containing lgG was significant for parietal cells only (P < 0.05), while that for rats injected with PCA and anti-human intrinsic factor (IF) antibodieswas significant for both parietal and peptic cells (P < 0.05). Difference between control rats injected with normal rabbit lgG and those injected with rabbit anti-rat intrinsic factor (IF) antibodies-containing lgG was significant both for peptic cells (P = 0.02) and parietal cells (P < ().()5).

402

INADA AND GLASS

When rabbit lgG containing both PCA and human IFA was given for the same length of time, a significant reduction (P < 0.05) of parietal and peptic cells occurred. Rats injected for 12 weeks with antihuman or anti-rat !FA-containing rabbit lgG showed reduction in the peptic cell mass by 23% and 38%, respectively, which was statistically significant for the anti-rat IFA only (P = 0.02). Administration of the rabbit IgG containing antibodies to rat IF also caused a statistically significant (P < 0.05) decrease of the parietal cell mass. In the groups of animals injected for 8 weeks only with antibodies-containing lgG the reduction in peptic and parietal cell masses was less pronounced. When the mean cellular masses were calculated per kg of weight of the rat, the animals receiving human !FA-containin g rabbit IgG showed marked reduction of peptic cell mass (by about ~0%), whereas those injected with rat IF A-containing rabbit IgG demonstrated a still larger decrease of the peptic cell mass by 30 to 35% (P < 0.05 and < 0.02, respectively). Effects of PCA- and IFA- containinR IgG's on HCl output and volume of gastric

I!JI 160

Vol. 69, No.2

secretion. The outputs of HCl after histamine during the last 2 weeks of a 12-week injection period were compared with that in the first 2 weeks of the saine injection period in s'even groups of 4 rats each. A ' significant reduction of the hourly HCl output from 36 ± 12 to 19 ± 12 ,uEq per hr, i.e., by 47% on the average was observed in rats injected with human lgG containing PCA. This decrease was in contrast with the mean increase by about 33% after 12 weeks of injection of saline or the normal human lgG, and by 45% after injections of the normal lgG (from 33 to 48 ,uEq per hr). When the output of HCl was calculated per kg of rat weight (fig. 4), the depressing effect of PCA-containing immunoglobulins on HCl output was also highly significant (reduction from 120 to 64 ,uEq per hr per kg, i.e., by 47%) (P < 0.01). This was in contrast with all other series of control rats as well as those injected with human or rabbit IgG contain ing IFA against human IF, where there was an increase in the HCl output after 12 weeks of injections, probably due to the growth of animals. The decrease of histamine-stimulated HCl output after 12 weeks of adm inistra-

moo

0-2 Weeks

10-12 Weeks Rabbit lgG

Human lgG

Controls

140

e

120

~ 100

5

0

L'

.....

80

0'

w 60 =>.

u ::r:

40 20 0

So line

Normal

PCA

PCA and antihuman IF

Normal

Anlihuman IF

Antirat IF

FIG. 4. H Cl output per kg of weight following daily administration to rats of IgG's of various sources or saline (means of 4 rats with standard error) . A highly significant (P < 0.01) reduction in HCl output following the 12 weeks of injection of lgG was observed in the group of rats receiving parietal cell antibodies (PCA).

August 1975

EFFECT OF /FA ON GASTRIC FUNCTION

tion of PCA or PCA- + IF A-containing IgG was associated with the 50% reduction in the volume of gastric secretion. A still larger and significant decrease of the volume of gastric secretion by 70% was found after administration of IgG from the sera of rabbits immunized with rat or human IF. Effects of PCA- and IFA-containint< l{!G on pepsin output. The administration of human IgG containing human PCA + IFA or rat IF A caused a decrease in the pepsin output by 52% on the average. This is shown by comparison of the mean pepsin output during the first 2 and the last 2 weeks of the 12-week injection period (P < 0.05). A similar decrease (P < 0.05) was observed in rats injected with rabbit lgG containing antibodies to rat IF, while all controls, including animals injected with normal human or rabbit lgG, showed either nonsignificant changes or an increase in the pepsin output . Nonsignificant changes were also noted in rats injected with rabbit lgG containing antibodies to human IF . The effects of antibodies on pepsin output are well illustrated in figure 5, where the mean pepsin outputs of rats injected daily for 12 weeks with IgG's from various sources or saline were compared with each other . Here, pepsin output was significantly lower in animals injected with rabbit IgG-containing antibodies to human or rat IF than in rats injected with normal rabbit lgG (P < 0.05 and < 0.02, respectively). These differences remained significant when pepsin output was calculated per hr and per kg of weight, as shown in figure 6. Here again the pepsin output was significantly reduced in rats injected with human IgG containing PCA or PCA + IFA to human IF (P < 0.01 and < 0.05, respectively) as well as in those injected with rabbit IgG containing antibodies to rat IF (P < 0.02). Rats injected with rabbit IgG containing anti-human IF A or normal IgG from human or rabbit serum showed a nonsignificant or none whatsoever reduction in pepsin output. Effects of PCA- and !FA-containing IgG on the IF output. When the IF outputs in rats injected with anti-human or anti-rat !FA-containing rabbit lgG's were com-

40:3

Sohne

Normal serum

PCA serum

PCAond onh-humon If mum

Normal serum

Anl1·humon If serum Ant~-rotlfserum

0

200 400 600 BOO 1000 Pepsin Equiv. pg I hour

1200

FIG . 5. Pepsin output in rats injected daily for I
pared with that observed in rats injected with saline or normal human or rabbit IgG they were found to be very markedly reduced to one-quarter to one-third of the control value (both P values < 0.01). A nonsignificant reduction was obtained in rats injected with lgG's from human sera containing PCA and ant ihuman IFA. It should be noted, however, that IgG from human sera containing PCA alone also caused reduction of the IF output (P < 0.02) when compared to rats injected with saline or IgG from normal human serum (fig. 7). When the hourly IF output was calculated per kg of rat weight (fig. 8) , its reduction in rats injected for 12 weeks with rabbit IgG containing antibodies to human and rat IF was still significant when compared to that injected with normal human IgG (P < 0.05). Pro{!ressiue effects on rat f
404

INADA AND GLASS

I!Jlo-2 Controls

4000

Vol . 69, No.2

rfttf@ ..... ...

Weeks

10-12 Weeks Rabbit lgG

Human lgG

3500

c;

~

I

3000

-""

'-

g

2500

..c:

'-

g:

2000

cr

LLl

c:

·v;

IT

1500

~

[l

0.

a.. "'

1000 500 0

Saline

Normal

PCA

PCA and antihuman IF

Normal

Antihuman IF

Antirat 1F

FIG. 6. P epsin output per kg of weight following daily administration to rats of lgG's of various sources or saline (means of 4 rats with standard error). Significant reductions in pepsin output during the 12 weeks of injection of lgG were observed in groups of rats receiving human lgG containing parietal cell antibodies (PCA) or PCA and anti-human intrinsic factor (IF) antibodies (P < 0.01 and < 0.05, respectively) and in rats receiving · rabbit lgG containing anti-rat !FA (P < 0.02) .

Saline

jected daily with normal rabbit IgG over a period of 12 weeks (fig. 10). This was probably due to progressive increase in the body weight of animals during that time (table 1).

·

Nor mol serum PCA serum

PCA and onlt·humon IF serum

Normal serum

Anti-human IF serum Anti-rat IF serum

0

10

20

30

40

50

ng 812 bound by l FI hour FIG. 7. Intrinsic factor (IF) output in rats injected daily for 12 weeks with lgG's of various sources or saline (means of 4 rats with standard error). Highly significant reduction in IF output was observed in rats receiving rabbit lgG containing anti-human or antirat IF antibodies as compared to those injected with normal rabbit lgG (P < 0.01) and in those receiving human lgG containing parietal cell antibodies (PCA) as compared to those injected with saline or normal human lgG (P < 0.02).

onset of injections. To the contrary, a gradual increase in the output of all secretory products of gastric glands, i.e., HCl, pepsin, and IF was observed in rats in-

Discussion The . mechanism of the effect of the humoral IF antibodies on peptic cells and their function is not clear. No evidence of cytotoxicity to peptic cells or cellular hypersensitivity reaction in gastric mucosa after prolonged administration of IF A has been observed. Many other possible mechanisms may be here entertained, yet their discussion will be omitted because of the lack of evidence. 'r:lJ.e..r~gl.,l_Ging effect of human and rabbit IF A to human IF on the nit peptic cell ·mass and outp~t of IF and pepsin was similar. However, rabbit antibodies to rat IF caused a greater reduction in t he rat peptic cell mass and pepsin and IF outputs than the rabbit antibodies to human IF. This may indicate some trait of species specificity of the IFA to rat IF . The cross-

August 1975

EFFECT OF !FA ON GASTRIC FUNCTJ()N

reactivity of rabbit antibodies to human and rat IF was manifest, however, when rabbit lgG containing IFA either to human or rat IF was reacted in the indirect Coon's test with normal rat gastric mucosa. Both antibodies produced immunofluorescence in the peptic cells of the rat, indicating an antigen-antibody reaction in rat peptic cells between the rat IF and the rabbit anti-rat and anti-human IFA. This also confirms the radioautographic evidence of Hoedemaker et al. 14 as to the origin of IF from peptic cells in the rat. The thinning of the gastric mucosa, reduction of the peptic cell mass, and Controls 0:

c!J

200

"'

'

0

2000- 50 -

1600- 40 -

~

z::0

50

"'cii N

g'

1200 ·- '1 0 -

Anti-

rat

j'

f', /

I

'o,....

,.\,.o'

800- 20-

/ ',

Pepsm , /

/ 'o

o---o'

0

2

4

6 8 WEEKS

10

12

HCL

decrease of the pepsin and IF outputs has to be considered as manifestation of' partial gastric atrophy. Analogous observations were reported in regard to parietal cell antibodies by Walder, 38 Tanaka and Glass, 20 and Kawashima, 39 who observed thinning of gastric mucosa and reduction of parietal cell mass and HCl output in dogs and rats following daily injection, for several weeks, of these antibodies. When the IgG fraction from PA patients containin'g both PCA and IFA was injected intravenously to rats in this stud'v for a prolonged time, this resulted in an additive effect of the twa antibodies o~U-.the parietal cell mass and the secretory output of HCl, and the other on the e tic eel mass an the secretory output of pepsin and IF'. However, in some of our rats the effect on the peptic cell mass and output of pepsin and IF was greater than when the IF A was injected singly. Moreover, we have confirmed here the previous-Obs-ervation from-OUTTaOoratoryioti1afl?rolongea- admif!-istration .of PCA to rats caused also a significant d~_<:..te.ase of the IF__puill_ut. --

0-

•

IF

800- 20-

0-

./

. ·- o······.o .. . ) .. o ······

Anti-

1200- 30-

10-

HCI

/

:

'0

0

Normal

ngB 1zih pEq /h

400-

..c.. ..

F!G . 10. Effect on gastric secretion of daily administration to rats of rabbit normalJgG (means of 4 ruts) . IF, intrinsic factor.

FIG. 8. Effect on intrinsic factor (IF) output per kg of weight of daily administration to rats of IgG's of various sources or saline (means of 4 rats with standard error). Significant reduction in the intrinsic factor output per kg of weight during the 12 weeks injection of IgG was observed in groups of rats injected with rabbit lgG containing a nti-human IF antibodies (P < 0.05), and anti-rat IF antibodies (P < O.O!i). 1F

/•-•\v:·•

400- - 10-

~~ human IF

Pepsin

•

0-2 weeks

0

0 Sohne

Eqpg/h

IF

- •

o · n ..

100

0

0

/•--..\._

·····-

.•·

:;:

...

HCI

( ] 10-12 weeks

_g 150

0

If

Eq)lg/h ngB 12th pEq/h

Rabbit lgG

,__ <(

Pepsm

405

0

2

4

6

8

ro

12

WEEKS

FIG . 9. Effect on gastric secretion of daily administration to rats of rabbit lgG containing antibodies to human and rat intrinsic factor (IF) (means of8 rats).

•

j

406

INADA AND GLASS

TAHLF. 1 .

Vol. 69, No .2

Mean wei!{hts (!{)with standard deviations of eiRht !{r oups of rats treated with I!JG's of va rious sourc es for 12 weeks

Group

Rat no.

1 2 3 4 5

12 12 12 12 12

6

12

7

12

8

12

Materials injected' None Saline Normal human IgG Normal ra bbit IgG PCA-containing hum an IgG PCA + IF A-cont a ining hum an IgG Hum an !FA-containin g rabbit IgG Rat IF A-containing rabbit IgG

At the

After 8 weeks of injection

onset

312 311 305 309 278

7 7 ± 6 ± 7 ± 12 ± ±

After 12 weeks of injection•

363 ± 330 ± 311 ± 321 ± 287 ±

18 16 22 20 20

371 ± 339 ± 323 ± 321 ± 298 ±

21 14 14 20 9

277 ±

7

271

±

25

260

±

25

276 ±

8

283

±

16

271

±

35

275

5

283

±

15

274

±

23

±

"These means have been calc ul ated from 8 rats. • PCA, parietal cell antibodies.

The plausible alternatives for the inter- dev~ment of the "idiopathic" atrophic pretation of this finding are: (1) the pres- gastritis and PAin man. ence of a .circulating antibody to peptic cells or pepsin in the ' PCA-contairiing REFERENCES serum of atrophic gastritis patients ; (2) the cellular nonspecificity of the PCA in the 1. Jeffri es GH , Hoskins DW, Sleisenger HM: Antibody to intrinsic fac tor in serum from rat; (3) the formation of an antigen-an'tipatients with pernicious anemia. J . Clin Invest body complex at the progenitor cells site, 41:1106-1115, 1962 from which both parietal and peptic cells 2. Abels J, Bouma W, Jansz A, et al: Experiments may be derived. No supportive evidence is on the intrinsic factor antibody in serum from available at this time for any of these patients with pernicious anemia. J Lab Clin Med explanations. 61:893-906, 1963 The participation of the humoral immu- 3. Te Velde K, Anders GJPA, Nieweg HO : Gastrite chronique et anticorps antiestomac. Rev Med nological mechanisms in production of gas10:1895- 1898, 1969 tric atrophy and secretory failure is 4. Fisher JM , Taylor KB: The significance of gastric strongly supported by the results of this antibodies. Br J Haematol 20:1-7, 1971 study, and the implications of this work for 5. Doniach D, Roitt IM, Taylor KB : Auto-immunity the natural history of gastric atrophy in in pernicious anemia and thyroiditis: a family man are self-explanatory. As ·could-be. exs tudy. Ann NY Acad Sci 124:605-625, 1965 pe~ted from a passive immunization 6. Irvine WJ, Davies SH, Teite lb aum S, et al: The method, the administration of humoral clinical and pathological significance of gastric antibodies to rats did not result in inflamparieta l cell antibody. Ann NY Acad Sci 124:657-691, 1965 m;rtory lesions of the gastnc mucosa and t he c'nanges produced could not be e~uated 7. Glass GBJ: Immune complexes in the gastric mucosa and their role in development of perniwith the histological picture of atrophic cious anemia and its precursor state (abstr). XIV gastntis in man. This reqnires active imInternational Congress of Hematology, 1972, mumzation with antigens contained io gasSao Pau lo, Brazil tric juice or gastric mucosa. 40 - 43 The work 8. Schwartz M: Intrinsic factor-inhibiting substance 44 of Krohn and Finlayson has recently in serum of orally treated patients with pernicious given an experimental support to the conanemia. Lancet 2:61-62, 1958 cept ventilated for a long time of the 9. Taylor KB : Inhibition of intrinsic factor by obligatory coexistence of both humoral and pernicious anemia sera. Lancet 2:106-108, 1959 cellular immunological reactions for the 10. Schade SG, Feick P, Muckerheide M, ct al:

August 1975

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13.

14.

15.

16.

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18.

19.

20.

21.

22.

EFFECT OF !FA ON GASTRIC FUNCTWN

Occurrence in gastric juice of antibody to a complex of intrinsic factor and vitamin 8 12 • N Eng! J Med 275:528-531, 1966 Bar-Shany S , Herbert V: Transplacentally acquired antibody of intrinsic factor with vitamin B, 2 deficiency. Blood 30:777-784, 1967 Fisher JM, Taylor KB: The intracellular localization of Castle's intrinsic factor by an immunofluorescent technique using autoantibodies. Immunology 16:779-784, 1969 Jacob E, Glass GBJ: Localization of intrinsic factor and complement fixing intrinsic factor-intrinsic factor antibody complex in parietal cell of man . Clin Exp Immunol 8:517-527, 1971 Hoedemaker PJ, Abels J , Watchers JJ, et al: Further investigations about the site of production of Castle's gastric intrinsic factor. Lab Invest 15:1163-1173, 1966 In ada M, Glass GBJ : Effects of prolonged administration of homologous and heterologous intrinsic factor antibodies to rats (abstr 368). Fourteenth Annual Meeting of the American Society of Hematology, San Francisco, California, December 1971 In ada M, Glass GBJ: Effects of prolonged administration of intrinsic factor antibodies on gastric morphology and secretion in rats (abstr). Fed Proc 31:299, 1972 In ada M, Glass GBJ: Gastric secretory failure caused by prolonged administration of antibodies to intrinsic factor (abstr). Gastroenterology 64:748, 1973 Glass GBJ , Tanaka N, Inada M: Gastric secretory failure in rats caused by prolonged administration of parietal cells antibodies and homologous or heterologous intrinsic factor antibodies. In Symposium on Gastric Secretory Inhibition Edited by J DeGraef, Ninth International Congress of Gastroenterology, Paris, July 1972 Glass GBJ: Endogenous inhibitors of gastric secretion. J . Earl Thomas Memorial Symposium at the Jefferson Medical College of Thomas Jefferson. Edited by MFH Friedman. Philadelphia, 1974 (in press) Tanaka N, Glass GBJ : Effect of prolonged administration of parietal cell antibodies from patients with atrophic gastritis and pernicious anemia on the parietal cell mass and hydrochloric acid output in rats. Gastroenterology 58:482-494, 1970 Klotz AP, Duvall ME: The laboratory determination of pepsin in gastric juice with radio-iodinated albumin. J Lab Clin Med 50:753, 1957 Agunod M, Yamaguchi N, Lopez R, et al: Correlative study of hydrochloric acid, pepsin and intrinsic factor secretion in newborns and infants.

407

Am .J Dig Dis 14:400- 414, l9GH 2:1. Hansen H.J , Miller ON , Gallo-Torres H. et al : Assay of intrinsic factor activity of human gastric juice with zirconium phosphate gel. Anal Biochem W:2H7- 2fJ:l, WG!i 24. Marks IN , Drysdale KM: A modification of Zimmermann 's method for differential staining of gastric mucosa. Stain Techno! :!2:48, l9fl7 25. Bralow Sl', Komarow SA: Parietal cell mass and distribution in stomachs of Wistar rats. Am ,] Physiol 20:l:Gf>O- GG2, l9!i2 26. Crean GP: Effect of hypophysectomy on the gastric mucosa of the rat. Gut: :l:l2, 1968 27. Weisberg H. Glass GH.J: A rapid quantitative method for measuring intestinal absorption of vitamin 8 12 in man using a double label hepatic uptake test. ,J Lab Clin Med GB:lii:l- 172, I!J66 28. Yamaguchi N, Glass GH.J: The determination of intrinsic factor in gastric secretory analysis. Ann NY Acad Sci 148:924- 944, l9G7 29. Ardeman S, Chanarin I: A method for the assay of human gastric intrinsic fitctor and for the detection and titration of antibodies against. intrinsic factor. Lancet. 2: 1:!1;0-1 :lG4, I!Jfi:l :10. Hansen H.J. Miller ON, Tan CN:· Assay of the auto-humoral antibody in pernicious anemia sera that neutralizes the vitamin 8 12 combining site of human intrinsic factor. Am ,J Clin Nutr 19: !0- !(), 1966 31. JacobE, Hansen HJ, Miller ON: Rapid assay of antibody (CAB) in serum of pernicious anemia patients, which reacts with intrinsic factorvitamin B 12 complex (abstr). Fed Proc 2G:4:l0, 1966 32. Yamaguchi N, Rosenthal WS, Glass GH.J: Study of the intestinal absorption of '"Cr-labeled intrinsic factor. Am ,J Clin Nutr 2:!: !.~G - 164 , 1970 33. Taylor KB, Roitt IM, Doniach D, et al: Auto-immune phenomena in pernicious anemia : gastric antibodies. Hr Med ,J 2: l:l47-l :lG2, I!J(i2 :34. Irvine WJ: Gastric antibodies studied by fluorescence microscopy. Q ,J Exp Physiol 58:427-4:!8, 19G:l 35. Vaerman JP, Heremans ,JF, Vaerman C: Studies of the immune globulins of human serum. I. A method for the simultaneous isolation of the three immune globulins ()'88, 'YIM and 'YIA) from individual small serum samples. J Immunol 91:7-10, 1963 36. Fiasse R, Brus I, Code CF, et al: Short term study of the effect of human parietal cell antibody on the secretion of hydrochloric acid in rats . Gut 10:39-44, 1969 37. Scheidegger, JJ: Une micro-methode de l'immunoelectrophorese. Int Arch Allergy 7:103llO, 1955 38. Walder AI: Experimental achlorhydria: techniques of production with parietal cell

408

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antibody. Surgery 64:175-184, 1968 39. Kawashima K: Effects of gastric antibodies on gastric secretion. 2. Effects of rabbit antibodies against rat gastric mucosa and gastric juice on gastric secretion in the rat. Jap J Pharmacol 22:155, 1972 40. Smith WO, Hoke R, Landy J, et al: The nature of the inhibitor effect of normal gastric juice on Heidenhein pouch dogs . Gastroenterology 34:181-187, 1958 41. Hennes AK, Sevelius H, Lewellyn T, et al: Atrophic gastritis in dog . Production by intradermal injection of gastric juice in Freund's

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adjuvant. Arch Pathol 73:281-287, 1962 42. Krohn K: Experimental gastritis in the dog. I. Production of atrophic gastritis and antibodies to parietal cells. Ann Med Exp Fenn 46:249-258, 1968 43. Fixa B, Kretz, Roemer GB: Chronische Gastritis. Eine immunobiologische Studie. Med Klin 64:2414-2419, 1969 44. Krohn KJE, Finlayson NDC: Interrelations of humoral and cellular immune responses in experimental canine gastritis . Clin Exp Immunol 14:237, 1973