Expression of Intrinsic Factor in Rat and Murine Gastric Mucosal Cell Lineages Is Modified by Inflammation
2000; Elsevier BV; Volume: 157; Issue: 4 Linguagem: Inglês
10.1016/s0002-9440(10)64635-4
ISSN1525-2191
AutoresJiansu Shao, R. Balfour Sartor, Elisabeth Dial, Lenard M. Lichtenberger, Wolfgang Schepp, David Alpers,
Tópico(s)IL-33, ST2, and ILC Pathways
ResumoIntrinsic factor is produced primarily by chief cells in rat and mouse, but 4 to 11% of isolated rat parietal cells also contain intrinsic factor. To test whether local conditions could alter the distribution of intrinsic factor expression, two rodent models of chronic lymphocytic gastric inflammation were examined. Immunocytochemistry was performed using antiserum against human intrinsic factor and H/K ATPase (a parietal cell marker), counting the percent of intrinsic factor-positive parietal cells. HLA-B27 transgenic rats develop chronic gastritis at age 3 months. Congenic controls expressed intrinsic factor in 8.9 ± 3.8% (mean ± SD) of parietal cells; in inflamed areas of transgenic rats 21 ± 5.2% (P < 0.0001) of parietal cells were positive. In adjacent areas without inflammatory infiltrate 16 ± 3.6% of parietal cells contained intrinsic factor. C57BL/6 mice inoculated with Helicobacter felis develop gastritis by 4 weeks. After 4 and 8 weeks of infection, intrinsic factor-positive parietal cells increased from 7.8 ± 2.8% in the congenic controls to 17.6 ± 4.1% in the inflamed gastric body (P < 0.0001). Isolated rat parietal cells incubated with interleukin-1β demonstrated a twofold increase in intrinsic factor-positive parietal cells. These studies are consistent with the concept that intrinsic factor expression is both predetermined in chief cells and can be expressed in parietal cells in response to local inflammatory factors. The differences between inflamed and adjacent noninflamed areas in the rat model suggest a tissue gradient of soluble inducer(s), possibly cytokines. Intrinsic factor is produced primarily by chief cells in rat and mouse, but 4 to 11% of isolated rat parietal cells also contain intrinsic factor. To test whether local conditions could alter the distribution of intrinsic factor expression, two rodent models of chronic lymphocytic gastric inflammation were examined. Immunocytochemistry was performed using antiserum against human intrinsic factor and H/K ATPase (a parietal cell marker), counting the percent of intrinsic factor-positive parietal cells. HLA-B27 transgenic rats develop chronic gastritis at age 3 months. Congenic controls expressed intrinsic factor in 8.9 ± 3.8% (mean ± SD) of parietal cells; in inflamed areas of transgenic rats 21 ± 5.2% (P < 0.0001) of parietal cells were positive. In adjacent areas without inflammatory infiltrate 16 ± 3.6% of parietal cells contained intrinsic factor. C57BL/6 mice inoculated with Helicobacter felis develop gastritis by 4 weeks. After 4 and 8 weeks of infection, intrinsic factor-positive parietal cells increased from 7.8 ± 2.8% in the congenic controls to 17.6 ± 4.1% in the inflamed gastric body (P < 0.0001). Isolated rat parietal cells incubated with interleukin-1β demonstrated a twofold increase in intrinsic factor-positive parietal cells. These studies are consistent with the concept that intrinsic factor expression is both predetermined in chief cells and can be expressed in parietal cells in response to local inflammatory factors. The differences between inflamed and adjacent noninflamed areas in the rat model suggest a tissue gradient of soluble inducer(s), possibly cytokines. Gastric glands in the zymogenic region of the stomach contain three major cell types: mucous surface cells, parietal cells, and zymogenic (chief) cells.1Karam SM Leblond CP Identifying and counting epithelial cell types in the “corpus” of the mouse stomach.Anat Rec. 1993; 236: 259-279Crossref PubMed Scopus (330) Google Scholar Intrinsic factor (IF) is a major secreted protein of the parietal cells in humans,2Glass GBJ Gastric Intrinsic Factor and Other Vitamin B12 Binders. Georg Thieme Publications, Stuttgart1974Google Scholar but in the mouse and rat IF is found in the zymogenic cells.3Boass A Wilson TH Cellular localization of gastric intrinsic factor in the rat.Am J Physiol. 1964; 206: 783-786PubMed Google Scholar, 4Lorenz RG Gordon JI Use of transgenic mice to study regulation of gene expression in the parietal cell lineage of gastric units.J Biol Chem. 1993; 268: 26559-26570Abstract Full Text PDF PubMed Google Scholar In human5Howard TA Misra DN Grove M Becich MJ Shao J-S Gordon MM Alpers DH Human gastric intrinsic factor expression is not restricted to parietal cells.J Anat. 1996; 189: 303-313PubMed Google Scholar and in rat gastric mucosa, IF is occasionally found inside cells of different lineage, especially in the rat in which 4 to 11% of isolated parietal cells express the protein.6Shao J-S Schepp W Alpers DH Expression of intrinsic factor and pepsinogen in the rat stomach identifies a subset of parietal cells.Am J Physiol. 1998; 274: G62-G70PubMed Google Scholar These IF-expressing parietal cells were distributed in the lower neck and basal regions of the gastric glands, consistent either with predetermination of expression within cell lineages, with parietal cell maturation, with induction of IF production by local tissue factors, or with a combination of these mechanisms. Local factors may account for the phenomenon of mosaicism noted in small intestinal epithelium.7Mauiri L Raia V Potter J Swallow D Ho MW Fiocca R Finzi G Cornaggia M Capella C Quaroni A Auricchio S Mosaic pattern of lactase expression by villous enterocytes in human adult-type hyplactasia.Gastroenterology. 1991; 100: 359-369PubMed Google Scholar, 8Rubin D Ong DE Gordon JI Cellular differentiation in the emerging fetal rat small intestinal epithelium: mosaic patterns of gene expression.Proc Natl Acad Sci USA. 1989; 86: 1278-1282Crossref PubMed Scopus (78) Google Scholar A number of cytokines, especially interleukin (IL)-1β and tumor necrosis factor (TNF)-α, can induce protein production in somatic cells.9Dinarello CA Biology of interleukin-1.FASEB J. 1988; 2: 108-115Crossref PubMed Scopus (1413) Google Scholar, 10Dayer JM Beutler B Cerami A Cachectin/tumor necrosis factor stimulates collagenase and PGE2 production by human synovial cells and dermal fibroblasts.J Exp Med. 1985; 162: 2163-2168Crossref PubMed Scopus (1096) Google Scholar Moreover, IL-1 receptors are found on the surface of parietal cells.11Schepp W Dehne K Herrmuth H Pfeffer K Prinz C Identification and functional importance of IL-1 receptors on rat parietal cells.Am J Physiol. 1998; 275: G1094-G1105PubMed Google Scholar Therefore, models of chronic inflammation of the zymogenic mucosa were selected to test whether increased elaboration of cytokines could enlarge the population of parietal cells that produce IF. HLA-B27, a serologically defined class 1 major histocompatibility complex allele, is associated with several human inflammatory disorders. An attempt was made to produce an animal model in transgenic mice, but despite physiologically normal function of B27 in hybrid mice, no features of human disease appeared.12Taurog JD Lowen L Forman J Hammer RE HLA-B27 in inbred and non-inbred transgenic mice. Cell surface expression and recognition as an alloantigen in the absence of human β2-microglobulin.J Immunol. 1988; 141: 4020-4023PubMed Google Scholar Because rats are susceptible to experimental arthritis that cannot be produced in mice, transgenic technology was developed in Fisher 344 and Lewis rats, and animals expressing HLA-B27 and human β2-microglobulin genes developed spontaneous multiorgan inflammatory disease.13Hammer RE Maika SD Richardson JA Tang J-P Taurog JD Spontaneous inflammatory disease in transgenic rats expressing HLA-B27 and human β2m: an animal model of HLA-B27-associated human disorders.Cell. 1990; 63: 1099-1112Abstract Full Text PDF PubMed Scopus (867) Google Scholar Two of the seven transgenic lines developed inflammation, including gastrointestinal disease and arthritis, increasing in severity from 5 to 15 weeks of age. F344 transgenic rats raised in a specific pathogen-free environment develop histological evidence of colitis and gastroduodenal inflammation by 10 to 12 weeks of age. No disease occurs in the absence of bacterial stimulation14Rath HC Herfarth HH Ikeda JS Grenther WB Hamm Jr, TE Balish E Taurog JD Hammer RE Wilson KH Sartor RB Normal luminal bacterial, especially Bacteroides species, mediate chronic colitis, gastritis, and arthritis in HLA-B27/human beta2 microglobulin transgenic rats.J Clin Invest. 1996; 98: 945-953Crossref PubMed Scopus (708) Google Scholar and disease correlates with cecal luminal anaerobic bacterial concentrations.15Rath HC Ikeda JS Linde H-J Schomerich J Wilson KH Sartor RB Varying cecal bacterial loads influences colitis and gastritis in HLA-B27 transgenic rats.Gastroenterology. 1999; 116: 310-319Abstract Full Text Full Text PDF PubMed Scopus (109) Google Scholar The incidence of disease varied from 30 to 100% in litters, and was affected by the genetic strain and density of animals/cage. The gastric body and antrum were involved with widely scattered mononuclear foci in the lamina propria and submucosa. The infiltrate occurred in some, but not all, crypt units, and was associated with a marked reduction in the number of parietal cells in some glands.13Hammer RE Maika SD Richardson JA Tang J-P Taurog JD Spontaneous inflammatory disease in transgenic rats expressing HLA-B27 and human β2m: an animal model of HLA-B27-associated human disorders.Cell. 1990; 63: 1099-1112Abstract Full Text PDF PubMed Scopus (867) Google Scholar Subsequently the model was reproduced in transgenic mice (mostly males), where it was associated with the presence of free heavy chains on a subpopulation of B27-expressing lymphocytes.16Khare SD Hansen J Harvinder SL David CS HLA-B27 heavy chains contribute to spontaneous inflammatory disease in B27/human β2-microglobulin (β2m) double transgenic mice with disrupted mouse β2m.J Clin Invest. 1996; 98: 2746-2755Crossref PubMed Scopus (116) Google Scholar In this model inflammation did not develop in germ-free animals, although no pathogen was detected in the gastrointestinal lumen. Helicobacter pylori infection causes clinical gastritis and peptic ulcers and is associated with the development of gastric cancer. Helicobacter felis is a spiral bacteria isolated from feline stomach that colonizes gastric mucosa in mice, rats, and dogs.17Lee A Fox JG Murphy J A small animal model of human Helicobacter pylori active gastritis.Gastroenterology. 1990; 99: 1315-1323Abstract PubMed Google Scholar H. felis-associated gastritis in mice reproduces many of the features of human disease, including a marked polymorphonuclear and mononuclear cell infiltrate.18Fox JG Blanco M Murphy JC Taylor NS Lee A Kabok Z Pappo J Local and systemic immune responses in murine Helicobacter felis active chronic gastritis.Infect Immun. 1993; 61: 2309-2315PubMed Google Scholar Production of H. felis infection in C57BL/6 mice was achieved by inoculating mice orally, and observing a similar infiltrate with a marked reduction in parietal cells noted by 6 months of infection.19Fox JG Li X Cahill RJ Andrutis K Rustgi AK Odze R Wang TC Hypertrophic gastropathy in Helicobacter felis-infected wild-type C57BL/6 mice and p53 hemizygous transgenic mice.Gastroenterology. 1996; 110: 155-166Abstract Full Text PDF PubMed Scopus (165) Google Scholar The organism colonizes the gastric mucous layer and causes gastritis in the antrum and body of the stomach by 4 weeks, with inflammation increasing further by 8 weeks after inoculation.20Lichtenberger LM Dial EJ Ottlecz A Romero JJ Lechago J Fox JG Attenuation of the hydrophobic phospholipid barrier is an early event in Helicobacter felis-induced gastritis in mice.Dig Dis Sci. 1999; 44: 108-115Crossref PubMed Scopus (27) Google Scholar These animal models were used to test the hypothesis that chronic inflammation could increase ectopic IF production in other gastric cell lineages, particularly the parietal cell that expresses IF in human mucosa. Despite the decrease in parietal cell populations that occurred during development of inflammation, both models demonstrated more than a twofold increase in the percent of parietal cells that expressed IF, but only when an inflammatory infiltrate was present in the tissue. Other gastric mucosal lineages did not express IF. These results are consistent with a cytokine-induced expression of IF in parietal cells, and suggest that IF expression in cells is not entirely predetermined, at least in certain subpopulations that might be potential precursors for zymogen cells. A colony of transgenic HLA-B27 rats and nontransgenic littermates were derived in a sterile environment at the University of North Carolina School of Medicine, populated with specific pathogen-free bacteria (documented Helicobacter sp. free), and maintained in that environment.15Rath HC Ikeda JS Linde H-J Schomerich J Wilson KH Sartor RB Varying cecal bacterial loads influences colitis and gastritis in HLA-B27 transgenic rats.Gastroenterology. 1999; 116: 310-319Abstract Full Text Full Text PDF PubMed Scopus (109) Google Scholar The colony, derived from specific pathogen-free inbred Lewis/CrlBR (LEW) rats, was originally obtained from Dr. Joel D. Taurog (Southwestern Medical School, Dallas, TX). Female C57BL/6 mice, purchased from Taconic Laboratories (Germantown, NY), were inoculated intragastrically three times on alternate days at 5 weeks of age with 1 × 109 cfu of H. felis (ATCC 49179). Transgenic and control rats were killed at 1, 2, or 3 months of age. H. felis inoculated mice were maintained for up to 4 weeks after infection.18Fox JG Blanco M Murphy JC Taylor NS Lee A Kabok Z Pappo J Local and systemic immune responses in murine Helicobacter felis active chronic gastritis.Infect Immun. 1993; 61: 2309-2315PubMed Google Scholar After rats or mice were euthanized with CO2 inhalation, the glandular rat stomachs were fixed in Bouin’s solution for 2 hours at room temperature, and transferred to 70% ethanol overnight at 4°C before embedding in paraffin. The body and antrum of the murine stomachs were fixed in 10% buffered formalin for a few hours, stored in 80% ethanol, and then embedded in paraffin. Sections were cut 4-μm thick and stained for hematoxylin and eosin (H&E), or used for immunocytochemistry. Inflammatory infiltrate was estimated as absent, moderate, or severe (0, +, or ++) by one observer (J-SS) in a blinded manner. These samples were obtained from the same animals reported previously.20Lichtenberger LM Dial EJ Ottlecz A Romero JJ Lechago J Fox JG Attenuation of the hydrophobic phospholipid barrier is an early event in Helicobacter felis-induced gastritis in mice.Dig Dis Sci. 1999; 44: 108-115Crossref PubMed Scopus (27) Google Scholar Immunostaining was performed using the standard avidin-biotin-peroxidase complex method as reported previously,6Shao J-S Schepp W Alpers DH Expression of intrinsic factor and pepsinogen in the rat stomach identifies a subset of parietal cells.Am J Physiol. 1998; 274: G62-G70PubMed Google Scholar and counterstained with hematoxylin. Endogenous peroxidase was quenched by pretreatment with 1% H2O2 in methanol for 20 minutes. The primary antiserum used was rabbit polyclonal anti-human IF (1:200), raised against recombinant human IF produced in baculovirus-infected Sf9 cells.5Howard TA Misra DN Grove M Becich MJ Shao J-S Gordon MM Alpers DH Human gastric intrinsic factor expression is not restricted to parietal cells.J Anat. 1996; 189: 303-313PubMed Google Scholar In some experiments recombinant purified human IF made in Pichia pastoris was added to the antiserum before adding to the section.21Wen J Kinnear MB Richardson MA Willetts NS Russell-Jones GJ Gordon MM Alpers DH Functional expression in Pichia pastoris of human and rat intrinsic factor.Biochim Biophys Acta. 2000; 1490: 43-53Crossref PubMed Scopus (13) Google Scholar Rabbit antibody against the β-subunit of rat H/K ATPase (amino acids 2 to 23) was obtained from Dr. Jeffrey I. Gordon (Washington University School of Medicine, St. Louis, MO). Second antibody was goat anti-rabbit IgG obtained from Vector Laboratories (Burlingame, CA). Parietal cells were identified by their large size, triangular shape, and different staining pattern compared with chief cells. Quantitation of parietal cells staining positively for IF was performed manually. All parietal cells in sections of rat and mouse stomachs were counted in eight ×200 magnified fields per section, prepared from four rats and three mice per group/time point. The mean ± SD values were compared by Student’s t-test of paired samples. Highly enriched preparations of parietal cells were isolated from rat gastric mucosa as described previously,6Shao J-S Schepp W Alpers DH Expression of intrinsic factor and pepsinogen in the rat stomach identifies a subset of parietal cells.Am J Physiol. 1998; 274: G62-G70PubMed Google Scholar and cultured for 24 to 48 hours.11Schepp W Dehne K Herrmuth H Pfeffer K Prinz C Identification and functional importance of IL-1 receptors on rat parietal cells.Am J Physiol. 1998; 275: G1094-G1105PubMed Google Scholar Enriched preparations of parietal cells were washed 3 times in serum-free culture medium, and cells were cultured for 48 hours in a 1:1 mixture of Ham’s F-12 medium/Dulbecco’s modified Eagle’s medium with HEPES plusl-glutamine without bicarbonate, and supplemented with insulin (5 μg/ml), transferrin (5 μg/ml), sodium selenite (5 ng/ml), hydrocortisone (4 mg/ml), epidermal growth factor (25 ng/ml), gentamicin (10 mg/100 ml), and bovine serum albumin (2 mg/ml).11Schepp W Dehne K Herrmuth H Pfeffer K Prinz C Identification and functional importance of IL-1 receptors on rat parietal cells.Am J Physiol. 1998; 275: G1094-G1105PubMed Google Scholar Thereafter, recombinant human IL-1β (2.5 pg/ml) or recombinant human interferon-γ (1.5 ng/ml) were added to fresh culture media without added growth factors for 4 hours at 37°C. The medium was aspirated, centrifuged, and stored at −20°C. Adherent cells were removed by trypsinization, washed in phosphate-buffered saline (PBS), centrifuged, and fixed in Bouin’s solution overnight before transfer to graded ethanol concentrations from 70 to 100%. The cells were embedded in paraffin and 5-μm sections of the cell pellet were prepared on slides, which were probed with antiserum against IF as described above. The cells on each slide were counted completely, each high-power field contained 108 to 384 parietal cells (total of 1773 to 4384 in each slide were counted), and the IF-positive cells recorded. The results were compared using a two-tailed Student’s t-test. Transgenic animals were studied at 1, 2, or 3 months of age. Congenic control animals were examined only at 3 months of age. In the gastric body of the control rats, 8.9% of the parietal cells were positive for IF (Table 1 and Figure 1). Figure 1 demonstrates the specificity of the antiserum used against IF. Neither normal rabbit serum (Figure 1B) nor anti-IF antiserum incubated with recombinant IF (Figure 1D)showed any reactivity in either chief cells or parietal cells.Table 1Percent of Parietal Cells Expressing Intrinsic Factor (IF) in HLA-B27 Transgenic RatsInflammationAnimal (n)Age (months)BodyAntrumParietal cells/magnified field (mean no. ± SD)IF-positive parietal cells (mean% ± SD)Controls (4)3−−110 ± 278.9 ± 3.8Transgenic (4)1−−122 ± 329.6 ± 5.6, P = 0.6Transgenic (4)2−+100 ± 239.2 ± 4.4, P = 0.7Transgenic (4)3++Non-inflamed areas116 ± 3816 ± 3.6, P < 0.0001*P by paired two-sample t test for means (with equal variance) of 3 month inflamed versus noninflamed areas. The gender of animals (male/female) was 0/4 for controls, 4/0 for 1-month-old transgenics, and 2/2 for 2- and 3-month-old transgenics.Inflamed areas77 ± 1921 ± 5.2, P < 0.0001*P by paired two-sample t test for means (with equal variance) of 3 month inflamed versus noninflamed areas. The gender of animals (male/female) was 0/4 for controls, 4/0 for 1-month-old transgenics, and 2/2 for 2- and 3-month-old transgenics.* P by paired two-sample t test for means (with equal variance) of 3 month inflamed versus noninflamed areas. The gender of animals (male/female) was 0/4 for controls, 4/0 for 1-month-old transgenics, and 2/2 for 2- and 3-month-old transgenics. Open table in a new tab The percent of positive cells in transgenic HLA-B27 rats at 1 and 2 months of age (9.6 and 9.2, respectively) was no different from that found in the gastric body of control rats (Table 1). There was no inflammation found in the body of these animals, although mild inflammation had developed in the antrum by 2 months of age. No parietal cells are found in the antrum of adult rats, however, and stains for IF were also negative. After 2 months the infiltrate in the gastric body was not uniform, involving some gastric glands, but not others (Figure 2A). By 3 months submucosal abscesses were found occasionally in the antrum, and in the body more extensive inflammation with dilation of the gastric pits (Figure 2B). This inflammation was mononuclear (Figure 2C). In control rats the IF-positive parietal cells were mostly located in the lower part of the crypt (Figure 2D). The findings in the 3-month-old transgenic rats were quite different. Chronic (lymphocytic) inflammation and mucosal hyperplasia was found in both the body and antrum of the stomach (Figure 2, A and B). The absolute number of parietal cells was decreased.17Lee A Fox JG Murphy J A small animal model of human Helicobacter pylori active gastritis.Gastroenterology. 1990; 99: 1315-1323Abstract PubMed Google Scholar Control rats had 110 ± 27 (mean ± SD) parietal cells per magnified field (n = 32), whereas inflamed areas in transgenic rats showed 77 ± 19 parietal cells (n = 32). In the inflamed glands, 21% of parietal cells were IF-positive. Most of the positive cells were found in the lower neck and basal regions of the glands, and none appeared to be dying or apoptotic cells. In adjacent glands that were not inflamed there were still 16% of parietal cells that were positive for IF (Figure 2, E and F, and Table 1). These values were significantly greater than that for the control rats. In addition, the inflamed glands and those adjacent to them were also different from each other (P < 0.0001). The identity of IF-positive cells with morphology-like parietal cells was confirmed using antiserum against H/K ATPase, a parietal cell marker. Figure 2G shows all parietal cells positive in a 3-month-infected transgenic animal. Figure 2H shows an adjacent section stained with antiserum against IF, demonstrating some cells positive for both markers (isolated arrows), and some parietal cells positive for H/K ATPase but negative for IF. After either 4 or 8 weeks of infection, the mucosa and the submucosa both showed inflammatory infiltration, although the degree and distribution of inflammation was somewhat variable. After 4 weeks the parietal cell density in some gastric glands was decreased, and scattered inflammatory cells were found in the mucosa of the gastric body (Figure 3, B compared with the control in A). The number of parietal cells seen in H&E sections was diminished by 8 weeks (see Figure 3C versusFigure 1, A and B).20Lichtenberger LM Dial EJ Ottlecz A Romero JJ Lechago J Fox JG Attenuation of the hydrophobic phospholipid barrier is an early event in Helicobacter felis-induced gastritis in mice.Dig Dis Sci. 1999; 44: 108-115Crossref PubMed Scopus (27) Google Scholar Control mice (n = six animals, 70 fields) showed 70 ± 29 parietal cells/magnified field compared with the value in infected mice (n = 6, 62 fields) of 50 ± 41, but no mucosal hyperplasia was noted as in the HLA-B27 rat model. Moreover, submucosal inflammation was present, and atrophic parietal cells were noted by their smaller size and diminished eosinophilic stain. These cells were seen mostly in the middle part of the gastric gland (Figure 3, C and E). Further analysis of these atrophic cells was made using antiserum against H/K ATPase. In the normal control mice, all parietal cells stained strongly positive (Figure 3D). In the 8-week-infected mice, on the other hand, the stain for H/K ATPase is much diminished compared with control tissues, and some cells that appear by morphology to be atrophic parietal cells are negative (Figure 3E). The percent of IF-positive cells doubled in the presence of inflammation (Table 2), and did not change from 4 to 8 weeks (P = 0.49). However, the degree of mucosal inflammation did not alter qualitatively either (compare Figure 3, B and C). Again, the increase in IF-positive cells was located primarily in the lower neck and basal regions of the gastric glands and these cells seemed normal morphologically (Figure 3, F–H).Table 2Percent of Parietal Cells Expressing Intrinsic Factor (IF) in H. felis-Infected MiceInflammationAnimal (n)Time (weeks)MucosaSubmucosaIF-positive parietal cells (mean % ± SD)Control (6)4/8−−7.8 ± 2.8H. felis (3)4+/+++17 ± 4.9, P < 0.0001*H. felis (3)8+/++++17.2 ± 3.2, P < 0.0001*H. felis (6)4/817.6 ± 4.1, P < 0.0001*All animals were females. *P value by paired two-sample t test for means of infected versus control animals. The P value comparing 4-week and 8-week infected animals was 0.49. Thus, these data were combined to provide an average value for all the infected animals (last line of table). Open table in a new tab All animals were females. *P value by paired two-sample t test for means of infected versus control animals. The P value comparing 4-week and 8-week infected animals was 0.49. Thus, these data were combined to provide an average value for all the infected animals (last line of table). The percent of IF-positive parietal cells incubated with the cytokine interleukin-1β increased from 3.14 ± 1.41% to 6.35 ± 4.05% (mean ± SD, P < 0.005, n = 17). On the other hand, incubation with interferon-γ produced a small decline in IF-reactive cells, from 1.83 ± 1.19% to 0.96 ± 0.85% (n = 10, P < 0.1). No difference in IF appearance in the medium was detected between control and cytokine-incubated cells. The findings in the control animals of both models confirms our earlier observations that IF was expressed in parietal cells of the gastric mucosa in rodents.6Shao J-S Schepp W Alpers DH Expression of intrinsic factor and pepsinogen in the rat stomach identifies a subset of parietal cells.Am J Physiol. 1998; 274: G62-G70PubMed Google Scholar The percent of IF-positive cells in the control groups was higher than found in outbred Sprague-Dawley rats (1.5 to 4%), but consistent with the percent of IF-positive parietal cells in isolated parietal cell preparations (4 to 11%).6Shao J-S Schepp W Alpers DH Expression of intrinsic factor and pepsinogen in the rat stomach identifies a subset of parietal cells.Am J Physiol. 1998; 274: G62-G70PubMed Google Scholar Previous studies of IF expression in mice using the same antiserum were unable to detect IF in parietal cells, although a different strain of mice was used, and the tissues were fixed for extended periods of time (usually days) before being analyzed.4Lorenz RG Gordon JI Use of transgenic mice to study regulation of gene expression in the parietal cell lineage of gastric units.J Biol Chem. 1993; 268: 26559-26570Abstract Full Text PDF PubMed Google Scholar It had been appreciated that IF is very sensitive to loss of antigenicity during fixation for electron microscopic findings,22Levine JS Nakane PK Allen RH Human intrinsic factor secretion: immunocytochemical demonstration of membrane associated vesicular transport in parietal cells.J Cell Biol. 1981; 90: 644-655Crossref PubMed Scopus (13) Google Scholar but it was not so clear that the same problem existed for light microscopic studies. Perhaps the small size of the murine tissue requires shorter fixation times. We routinely use only a few hours for fixation of gastric tissue before immunolocalization studies and find that time quite sufficient for adequate tissue preservation. The finding of ectopic production of IF, especially located in the lower neck and basal regions of the gastric gland, suggests that the expression of chief cell products in parietal cells is either hard-wired (predetermined by lineage or by functional maturation), or is influenced by environmental factors. The increase in IF-positive parietal cells in two models of chronic inflammation of the gastric body suggests that local environmental factors may be important. The rat model demonstrates gastric mucosal hyperplasia, whereas in the mouse model patches of atrophic mucosa are seen without intervening hyperplasia. Both models cause a marked decrease in the total number of parietal cells, and some of the remaining parietal cells in the middle of the murine gastric glands were atrophic. It seems unlikely that IF expression is solely a measure reflecting cell damage, as none of the IF-positive cells in or near the base of the gastric glands appeared abnormal morphologically. In addition, H. felis infection of C57BL6 mice show a marked diminution in chief cell number with a decrease in IF immunostaining.23Wang TC Goldenring JR Dangler C Ito S Mueller A Jeon WK Koh TJ Fox JG Mice lacking secretory phospholipase A2 show altered apoptosis and differentiation with Helicobacter felis infection.Gastroenterology. 1998; 114: 675-689Abstract Full Text Full Text PDF PubMed Scopus (208) Google Scholar It is possible that the proliferative zone containing relatively undifferentiated cells is expanded whether mucosal hyperplasia or atrophy is present. For example, in the presence of inflammation the parietal cell precursors capable of IF expression (eg, preneck precursor lineage in the mouse,1Karam SM Leblond CP Identifying and counting epithelial cell types in the “corpus” of the mouse stomach.Anat Rec. 1993; 236: 259-279Crossref PubMed Scopus (330) Google Scholar might be preferentially preserved, creating a falsely elevated percent of IF-positive cells. However, in relatively normal gastric glands adjacent to the inflamed ones (in the rat model) the percent of IF-positive cells was also higher, although the number of parietal cells seemed normal. Thus, it seems unlikely that preservation of an IF-expressing lineage accounts for the observed changes. The most likely explanation for these findings induced by inflammation is that they are related to recruitment of parietal cells newly expressing IF. The IF-positivity was present only when the gastric body is inflamed and the percent of positive cells was higher in inflamed areas than in noninflamed glands, at least in the rat model that allows such comparisons. Moreover, the difference between inflamed and noninflamed areas suggests a gradient effect, consistent with the presence of a diffusible inducer, perhaps secreted by (or liberated from) infiltrating inflammatory cells. The inflammation might be related either to a primary effect on the mucosa or a secondary one mediated by microbial presence and stimulation.15Rath HC Ikeda JS Linde H-J Schomerich J Wilson KH Sartor RB Varying cecal bacterial loads influences colitis and gastritis in HLA-B27 transgenic rats.Gastroenterology. 1999; 116: 310-319Abstract Full Text Full Text PDF PubMed Scopus (109) Google Scholar The cytokines produced by inflammatory cells that have been often associated with increased protein expression are IL-1, TNF-α, and interferon-γ. The mRNAs encoding these cytokines (among others) are abundantly expressed in the gastric mucosa of patients with chronic gastritis,24Ishihara S Fukuda R Fukumoto S Cytokine gene expression in the gastric mucosa: its role in chronic gastritis.J Gastroenterol. 1996; 31: 485-490Crossref PubMed Scopus (20) Google Scholar and expression is increased in the inflamed colons of HLA-B27 transgenic rats.15Rath HC Ikeda JS Linde H-J Schomerich J Wilson KH Sartor RB Varying cecal bacterial loads influences colitis and gastritis in HLA-B27 transgenic rats.Gastroenterology. 1999; 116: 310-319Abstract Full Text Full Text PDF PubMed Scopus (109) Google Scholar Gastric parietal cells have receptors for IL-1 at least, and this cytokine has been demonstrated to have antisecretory activity.11Schepp W Dehne K Herrmuth H Pfeffer K Prinz C Identification and functional importance of IL-1 receptors on rat parietal cells.Am J Physiol. 1998; 275: G1094-G1105PubMed Google Scholar, 25Tache Y Saperas E Potent inhibition of gastric acid secretion and ulcer formation by centrally and peripherally administered interleukin-1.Ann NY Acad Sci. 1992; 664: 353-368Crossref PubMed Scopus (52) Google Scholar IL-10 and TNF-α production26Bodger K Wyatt JI Heatley RV Gastric mucosal secretion of interleukin-10: relations to histopathology, Helicobacter pylori status, and tumour necrosis factor-alpha secretion.Gut. 1997; 40: 739-744Crossref PubMed Scopus (98) Google Scholar and IL-1β, IL-6, IL-8, and IL-12 as well,27Bauditz J Ortner M Bierbaum M Niedobitek G Lochs H Schreiber S Production of IL-12 in gastritis relates to infection with Helicobacter pylori.Clin Exp Immunol. 1999; 117: 316-323Crossref PubMed Scopus (54) Google Scholar are increased in antral mucosal biopsies from patients with chronic gastritis, and the cytokine secretion varies with the degree of inflammation.26Bodger K Wyatt JI Heatley RV Gastric mucosal secretion of interleukin-10: relations to histopathology, Helicobacter pylori status, and tumour necrosis factor-alpha secretion.Gut. 1997; 40: 739-744Crossref PubMed Scopus (98) Google Scholar IL-8 and TNF-α production were increased in patients with H. pylori infection in one study,28Fan XG Chua A Fan XJ Keeling PW Increased gastric production of interleukin-8 and tumour necrosis factor in patients with Helicobacter pylori infection.J Clin Pathol. 1995; 48: 133-136Crossref PubMed Scopus (135) Google Scholar but not in another.27Bauditz J Ortner M Bierbaum M Niedobitek G Lochs H Schreiber S Production of IL-12 in gastritis relates to infection with Helicobacter pylori.Clin Exp Immunol. 1999; 117: 316-323Crossref PubMed Scopus (54) Google Scholar The studies with isolated parietal cells showed that at least one cytokine that contributes to a Th1 response (IL-1β) reproduced in vitro the twofold increase in IF-positivity seen in vivo. Atrophy of parietal and zymogenic cells is an eventual outcome of chronic inflammation of the gastric body, but dysplasia precedes the atrophy in most cases. The altered IF expression might be an early marker of such dysplastic changes. It is possible that the observed increase in IF immunoreactivity is related to a decrease in IF secretion, as IF and H+ secretion occur by the same canalicular pathway.22Levine JS Nakane PK Allen RH Human intrinsic factor secretion: immunocytochemical demonstration of membrane associated vesicular transport in parietal cells.J Cell Biol. 1981; 90: 644-655Crossref PubMed Scopus (13) Google Scholar For this explanation to be valid, it would be necessary for some parietal cells to be producing undetectable IF under basal conditions. Alternatively, the cytokines released during inflammation could up-regulate expression of IF in cells which constitutively express undetectable amounts, recruit new cells to produce IF, expand a population of parietal cells that normally produce IF, or have combined effects. The results with isolated parietal cells favor up-regulation of IF expression. This study did not evaluate the quantitative secretion of IF from chief cells, although there was no obvious change in their immunoreactivity (Figure 2, Figure 3). IL-1β, on the other hand, does not affect basal or stimulated pepsinogen secretion from chief cells.29Serrano JT Lanas AI Lorente S Sainz R Cytokine effects on pepsinogen secretion from human peptic cells.Gut. 1997; 40: 42-48PubMed Google Scholar Whatever the mechanism whereby cytokines may produce the effect noted on parietal cells, it is possible that increased IF-positivity represents a surrogate marker for the local effect of one or more cytokines. If so, this would be very instructive in following the tissue effects of cytokine stimulation and in evaluating this response as a marker for eventual mucosal atrophy. We thank Feng-Ling Li for providing excellent technical support for the HLA-B27 studies.
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