Journal of Clinical Pathology, 1978, 31, 578-584

Ontogeny of actin and microsomal antigens ingastric parietal cellsR. CEREDIG1 AND B. H. TOH

From the Department ofPathology and Immunology, Monash University Medical School,Commercial Road, Prahran, Victoria 3181, Australia

SUMMARY Six fetal and 10 neonatal rat or mouse stomachs and a 14-week human fetal stomachwere examined for immunofluorescence reactivity with four sera containing parietal cell antibody(PCA) and four other sera containing smooth muscle antibody (SMA). In rat and mouse stomachs,parietal cells first reacted with PCA in 19-day fetal stomachs and with SMA in two-day neonatalrat stomachs or newly-born mouse stomachs. SMA reactivity with fetal rodent stomachs wasrestricted to the cytoplasm of smooth muscle, the apices of gastric mucosal cells, and the cytoplasmof fibroblasts surrounding invaginating gastric pits. In the 14-week human fetal stomach, parietal cellsstained with PCA but not with SMA. Specificity of the staining reactions was established by thecomplete inhibition of PCA staining by serum absorption with a gastric microsomal fraction butnot with actin. Conversely, the SMA staining was abolished by serum immunoabsorption with actinbut not with the microsomal fraction. These observations, indicating that the development of theparietal cell microsomal antigen precedes that of actin, may be used to distinguish between thestaining of parietal cells by SMA and PCA.

In a previous study (Ceredig and Toh, 1977), wereported that smooth muscle autoantibody (SMA)present in the sera of some patients with activechronic hepatitis (ACH) gave immunofluorescentstaining of gastric parietal cells in a pattern indis-tinguishable from that obtained with parietal cellautoantibody (PCA). Furthermore, we also showedthat this SMA reaction with parietal cells could becompletely inhibited by serum absorption withskeletal muscle actin but not by a microsomal frac-tion derived from the gastric mucosa. Confirmationof the presence of actin in parietal cells has now beenprovided by the observation that experimentallyproduced anti-actin antibody reacts with these cells(Li et al., 1977). Also, ultrastructural studies usingheavy meromyosin have shown that actin-like micro-filaments are closely associated with the secretarycanalicular system of parietal cells and that, duringacid secretion, there is fusion of the membranes ofthe canalicular system and those of the apical tubulo-vesicular system (Vial and Garrido, 1976). Vial andGarrido (1976) suggested that actin filaments mayplay an active role in this intermembrane fusion.'Present address: Walter and Eliza Hall Institute, PostOffice, Royal Melbourne Hospital, Victoria 3050,Australia.Received for publication 10 October 1977

Our demonstration ofSMA reactivity with parietalcells indicates that a confident diagnosis of PCAcannot be made on routine immunofluorescencetests using sections of adult stomach when SMA isalso present in the same serum (Ceredig and Toh,1977). The present study on the ontogeny of actinand microsomal antigens in gastric parietal cells wasundertaken in an attempt to resolve this diagnosticproblem.

Material and methods

PATIENTS SERAFour PCA and four SMA sera were used in thisstudy. The four PCA sera, obtained from patientswith pernicious anaemia, had staining titres of > 32for adult mouse stomach. The PCA sera were alsocharacterised by specific reactivity with parietal cellsin stomachs derived from the adult rat, human, ox,rabbit, and guinea pig.The four SMA sera obtained from patients with

ACH were characterised by reactivity with smoothmuscle cytoplasm (Johnson et al., 1965), skeletalmuscle striations (Toh et al., 1978), hepatocytes ina 'polygonal' pattern (Farrow et al., 1971), renalglomeruli, thymus medulla (Whittingham et al.,1966), parietal cells (Ceredig and Toh, 1977), and

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central nervous system synapses (Toh et al., 1976).The sera had staining titres of > 64 for smoothmuscle, and > 8 for adult parietal cells (Ceredig andToh, 1977).

All sera were used fresh or stored at -30C forup to three years when they were rapidly thawed ina 37°C water-bath and tested at an initial dilutionof 118.

GASTRIC TISSUESFetal and neonatal stomachs were obtained frompregnant Sprague-Dawley rats (weight 300 g). Fetalrat stomachs were obtained from embryos at 14, 16,18, 19, 20, and 21 days of gestation. Neonatal ratstomachs were obtained from rats at 0, 1, 2, 3, 4, 5,7, 10, 14, and 21 days after birth. A parallel studyusing stomachs from fetal and neonatal BALB/cmice and a stomach from a 14-week human fetuswas also carried out.

Specimens of the above tissues were snap-frozenin an isopentane-liquid nitrogen slurry at - 170°C,and cryostat sections were examined for reactivitywith SMA or PCA.Comparable specimens of the above tissues were

also fixed in 10% buffered formalin, and 6 gmparaffin sections were stained with haematoxylin andeosin, Masson's trichrome, or a modified Zimmer-man stain for parietal cells (Irvine, 1963).

IMMUNOHISTOLOGYCryostat sections of 6 ,um of fetal and neonatalstomachs were examined by indirect immuno-fluorescence (Nairn, 1976) with SMA or PCA. Allstomachs were sectioned longitudinally so thatrepresentative areas of cardia, fundus, and antrumwere present in the same section. The conjugate forimmunofluorescent tracing of bound immuno-globulin was a fluorescein-isothiocyanate-labelledgoat anti-human-gamma-globulin with a fluoresceinto protein molar ratio of 4.0 and a protein contentof 0.8 g/100 ml. After immunofluorescent staining,the microscopical sections were examined by dark-ground ultraviolet fluorescent microscopy using acondenser fitted with a toric lens and a colourlessbarrier filter.

Specificity tests were carried out by immuno-absorption of SMA or PCA sera with skeletalmuscle G-actin or a bovine microsomal fractionderived from the gastric mucosa (Ceredig and Toh,1977).

Results

FETAL RAT STOMACHSPrimitive parietal cells, characterised by a slightlypaler eosinophilic cytoplasm, were first seen as

occasional cells at the bases of gastric pits of 19-dayfetal rat stomachs. At this stage, the primitive gastricpits were seen as invaginations of the relativelyundifferentiated epithelium into the deeper tissues;at the points of invagination these gastric pits weresurrounded by a collar of fibroblastic cells (Fig. 1).The primitive parietal cells did not stain with themodified Zimmerman stain.

Cryostat sections of rat stomachs obtained fromfetuses at 14, 16, and 18 days of gestation gavenegative staining reactions with PCA. However, in19-day fetal rat stomachs, weak cytoplasmic stainingof primitive parietal cells by PCA was seen (Fig. 2).

Parallel cryostat sections of fetal rat stomachsreacted with SMA gave staining of the smoothmuscle of the muscularis externa, the apices ofoccasional gastric mucosal cells, and the cytoplasmof fibroblasts surrounding invaginated gastric pits(Fig. 3). The cytoplasm of mucosal cells, includingthose of primitive parietal cells, did not stain.

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Fig. 1 Nineteen-day fetal rat stomach showinginvagination of relatively undifferentiated mucosalepithelium into the deeper tissues. A collar offibroblasticcells surrounds the margins of the invaginated gastric pits.Haematoxylin and eosin x 250.

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Fig. 3 Similar field to that in Fig. 1, reacted withSMA, showing staining of the muscularis externa, theapices ofgastric mucosal cells, and the cytoplasm offibroblasts surrounding invaginated gastric pits. Indirectimmunofluorescence x 200.

Fig. 2 Nineteen-day fetal rat stomach,reacted with PCA, showing cytoplasmicstaining ofa primitive parietal cell.Indirect immunofluorescence x 500.

RAT STOMACHS AT BIRTHAt birth, the mucosa had developed and differentiatedconsiderably with an increase in number and size ofgastric glands; parietal cells with an eccentric nucleusand pale eosinophilic cytoplasm were clearly seen(Fig. 4). A distinct muscularis mucosae, comprisingseveral cell layers, was also present. The parietalcells reacted positively with the modified Zimmermanstain.

Cryostat sections of these stomachs gave granularcytoplasmic fluorescence of parietal cells whenreacted with PCA (Fig. 5). The cytoplasm of thesecells did not react with SMA; positive SMA stainingwas restricted to the apices of mucosal cells, themuscularis mucosae (Fig. 6), and the muscularisexterna.

NEONATAL RAT STOMACHSFurther maturation of the gastric mucosa occurredin the first week after birth. At 6 days after birth(Fig. 7) the glands had increased in depth, the gastricpits were lined by predominantly mucus-producingcells, the mucosa was considerably folded with rugaeformation, the muscularis mucosae projected asvillous cores between gastric glands, and clearlyidentifiable parietal cells were presented; the latterreacted positively with the modified Zimmermanstain.

In cryostat sections of 2-day-old neonatal ratstomachs, occasional parietal cells gave weak stain-ing with SMA. In 6-day-old stomachs, parietal cellsreacted with PCA (Fig. 8) and SMA (Fig. 9) inpatterns identical with those seen in adult rat

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Fig. 5 Similar field to that in Fig. 4, reacted withPC4, showing granular cytoplasmic fluorescence ofparietal cells. Indirect iinmunofluorescence x 200.

Fig. 4 Rat stomach at birth showing numerous gastricglands. Parietal cells with an eccentric nucleus and palecytoplasm are clearly seen. H & E x 320.

stomachs. The intensity of SMA staining of parietalcells was greater in those parietal cells at the basesthan in those at the neck of glands.

FETAL AND/OR NEONATAL MOUSE AND HUMANSTOMACHSA similar study carried out on fetal and neonatalmouse stomach showed that the earliest reaction ofparietal cells with PCA was seen in stomachs from19-day-old fetuses while that with SMA was firstseen in stomachs from newly born mice (22 days).

Cryostat sections of human stomach obtainedfrom a 14-week fetus showed that parietal cellsstained with PCA (Fig. 10) but not with SMA; thelatter autoantibody reacted only with smooth muscle(Fig. 11).

SERUM TITRATIONSThe results of titrations of one PCA (76/2776) andone SMA (74/2001) serum against sections of fetal

Fig. 6 Similar field to that in Fig. 4, reacted withSMA, showing staining of the apices of mucosal cellsand the muscularis mucosae. Indirect immunofluorescencex 200.

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Fig. 9 Similar field to that in Fig. 7, reacted withSMA, showing staining of the muscularis mucosae and thecytoplasm ofparietal cells. Indirect immunofluorescencex 200.

Fig. 7 Six-day neonatal rat stomach, showing deepgastric pits lined predominantly by mucus-producingcells. Clearly identifiable parietal cells are present.H&E x 250.

Fig. 8 Similar field to that in Fig. 7, reacted withPCA, showing staining of the cytoplasm ofparietal cells.Indirect immunofluorescence x 200.

Fig. 10 Fourteen-week human fetal stomach, reactedwith PCA, showing staining ofParietalcells. (The muscularisdid not stain.) Indirect immunofluorescence x 320.

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Fig. 11 Similar field to that in Fig. 10, reacted withSMA, showing staining of the muscularis externa.Parietal cells do not stain. Indirect immunofluorescencex 320.

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14 16 18 20 22 2 4 6 8 10 12 14(Foetal) (Neonatal) (Adult)

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Fig. 12 Results of titrations ofPCA and SM.4 againstrat parietal cells (PC).

and neonatal rat stomachs are shown in Figure 12.The PCA serum, with a staining titre of 256 for adultrat parietal cells, gave a titre of 8 for parietal cells instomachs from 19-day-old fetuses; adult titres wereattained 14 days after birth. The SMA serum, with atitre of 256 for fetal and adult smooth muscle and 32for adult parietal cells, gave a titre of 8 for parietalcells in 2-day-old neonatal rat stomachs; adult titreswere obtained with 6-day-old neonatal stomachs.

SPECIFICITY STUDIES

Immunoabsorption experiments showed that thestaining of parietal cells by PCA was completely

inhibited by serum absorption with a gastric micro-somal fraction from bovine stomach but not byskeletal muscle G-actin. Conversely, the staining ofparietal cells by SMA was completely inhibited byserum absorption with G-actin but not by the gastricmicrosomal fraction.

Discussion

The results of our studies indicate that the develop-ment of gastric parietal cell (microsomal) auto-antigen and that of parietal cell actin are completelydissociated in the rat, mouse, and human stomachs.In all three species, the development of the gastricmicrosomal antigen preceded that of actin.The demonstration of the gastric microsomal

antigen by PCA in rat gastric parietal cells at 19 daysof gestation correlates well with light and electronmicroscopic identification of parietal cells at thisstage of development (Helander, 1969). Also, Hoede-maeker and Ito (1970), using the immunoperoxidaselabelling technique, have ultrastructurally localisedPCA staining to the membranes of the secretorycanalicular system; and this membrane system iswell developed in 19-day fetal rat stomachs(Helander, 1969). Furthermore, although lack ofmaterial prevented a complete developmental studyin the human, demonstration of the parietal cellautoantigen in the 14-week fetal human stomach isconsistent with the report that identifiable parietalcells have already developed at 11 weeks of gestation(Nomura, 1966). The results suggest that PCA maybe used as a marker for morphologically developedparietal cells.

Previous studies have reported that morphologicalmaturation of parietal cells occurs before functionalmaturation (Helander, 1969). For instance, whereasparietal cells are morphologically identifiable by day19 of gestation in the mouse, acid secretion does notoccur until four days after birth (Deren, 1971). Also,in the human, while parietal cells are first identifiedhistologically at 11 weeks' gestation, acid secretionbegins only at 16 weeks (Nomura, 1966). We andothers (Vial and Garrido, 1976) have postulated thatactin-like microfilaments in parietal cells may playa role in acid secretion. Our demonstration that thedevelopment of actin in parietal cells occurs afterthat of the microsomal antigen but before the onsetof acid secretion is consistent with this view.The luminal staining of gastric mucosal cells by

SMA may represent actin-like microfilaments. Thelatter have been identified ultrastructurally in similarapical locations in other developing glands (Wessellset al., 1977). Wessells et al. (1977) have suggestedthat gland formation by invagination of mucosalepithelium may occur as a result of contraction of

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microfilament 'purse-strings'. We suggest that thepresence of actin in these apical sites in mucosal cellsmay play a similar role in gastric gland formation.This suggestion is supported by the demonstrationthat cytochalasin B, which reversibly disrupts micro-filaments, specifically inhibits corticosteroid-inducedrat gastric mucosal maturation and differentiation(Yeomans et al., 1976).The fibroblasts surrounding gastric pits may

represent a mesenchymal reaction to the invaginatinggland. These cells stained positively with SMA. Theyprobably correspond to 'myofibroblasts' previouslydescribed in healing wounds (Gabbiani et al., 1972).Similar myofibroblastic reaction to invading tumourshas also been reported (Toh et al., 1977).

Finally, the demonstration that the expression ofactin and of microsomal antigens are dissociated inthe developing stomach provides a means of dis-tinguishing parietal cell staining by PCA and SMA.We recommend that, in clinical situations wherethere is staining of both smooth muscle as well asparietal cells, sections of stomachs of new-born ratsshould be tested with the serum to determine whetherthere are one or two autoantibodies present.

We thank Miss Vivien Randell for technicalassistance, Dr H. A. Ward for the gastric microsomalfraction, and Dr F. M. Clarke for the skeletalmuscle G-actin. This work was supported by grantsfrom the Anti-Cancer Council and the NationalHealth and Medical Research Council of Australia.

References

Ceredig, R., and Toh, B. H. (1977). Reaction of humansmooth muscle autoantibody with gastric parietal cells:a pitfall in the diagnosis of parietal cell autoantibody.Journal of Clinical Pathology, 30, 666-670.

Deren, J. S. (1971). Development of structure andfunction in the fetal and newborn stomach. AmericanJournal of Clinical Nutrition, 24, 144-159.

Farrow, L. J., Holborow, E. J., and Brighton, W. D.(1971). Reaction of human smooth muscle antibodywith liver cells. Nature, New Biology, 232, 186-187.

Gabbiani, G., Hirschel, B. J., Ryan, G. B., Statkov,P. R., and Majno, G. (1972). Granulation tissue as acontractile organ. A study of structure and function.

Journal of Experimental Medicine, 135, 719-734.Helander, H. F. (1969). Ultrastructure and function of

gastric parietal cells in the rat during development.Gastroenterology, 56, 35-52.

Hoedemaeker, P. J., and Ito, S. (1970). Ultrastructurallocalization of gastric parietal cell antigen withperoxidase-coupled antibody. Laboratory Investigation,22, 184-188.

Irvine, W. J. (1963). Gastric antibodies studied byfluorescence microscopy. Quarterly Journal of Experi-mental Physiology, 48, 427-438.

Johnson, G. D., Holborow, E. J., and Glynn, L. E. (1965).Antibody to smooth muscle in patients with liverdisease. Lancet, 2, 878-879.

Li, A. K. C., Trenchev, P. S., Holborow, E. J., Newsome,C., and Wynne, A. T. (1977). Experimental smoothmuscle antibodies. Clinical and Experimental Immuno-logy, 27, 273-277.

Nairn, R. C. (1976). Fluorescent Protein Tracing, 4thedition, Churchill Livingstone, Edinburgh and London.

Nomura, Y. (1966). On the submicroscopic morpho-genesis of parietal cell in the gastric gland of thehuman foetus. Zeitschrift fur Anatomie und Entwick-lungsgeschichte, 125, 316-356.

Toh, B. H., Cauchi, M. N., Cook, P. C., and Muller,H. K. (1977). Increased expression of actin-likecontractile protein in preneoplastic and neoplasticlesions in rat liver. British Journal of Cancer, 35,761-767.

Toh, B. H., Clarke, F. M., and Ceredig, R. (1978).Reaction of human smooth muscle autoantibody withskeletal muscle, cardiac muscle and thymic nyoid cells.Clinical Immunology and Immunopathology, 9, 28-36.

Toh, B. H., Gallichio, H. A., Jeffrey, P. L., Livett, B. G.,Muller, H. K., Cauchi, M. N., and Clarke, F. M. (1976).Anti-actin stains synapses. Nature, 264, 648-650.

Vial, J. D., and Garrido, J. (1976). Actin-like filamentsand membrane rearrangements in oxyntic cells.Proceedings of the National Academy of Sciences of theUnited States of America, 73, 4032-4036.

Wessells, N. K., Spooner, B. S., Ash, J. F., Bradley,M. 0., Luduena, M. A., Taylor, E. L., Wrenn, J. T.,and Yamada, K. M. (1977). Microfilaments in cellularand developmental processes. Science, 171, 135-143.

Whittingham, S., Mackay, I. R., and Irwin, J. (1966).Autoimmune hepatitis: immunofluorescence reactionswith cytoplasm of smooth muscle and renal glomerularcells. Lancet, 1, 1333-1335.

Yeomans,N. D., Trier, J. S., Moxey, P. C., and Markezin,E. T. (1976). Maturation and differentiation of culturedfetal stomach. Gastroenterology, 71, 770-777.

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