ARTICLE |
Correspondence to: Bradley A. Schulte, Dept. of Pathology and Laboratory Medicine, Medical U. of South Carolina, 165 Ashley Avenue, Suite 309, PO Box 250908, Charleston, SC 29425. E-mail: schulteb@musc.edu
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Summary |
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The NF-B/I
B complex is a major transcription regulator of inflammatory and immune responses. Helicobacter pylori infection causes chronic inflammation in gastric mucosa by inducing dissociation of the inhibitory I
B protein from the complex with a resulting increased expression of interleukin (IL)-8. To clarify which of several known I
B proteins could be involved in this inflammatory response, we undertook immunohistochemical examination of normal mouse stomach as well as other murine tissues for comparison, using polyclonal antibodies specific for
-, ß-,
-, and
-isoforms of I
B. The results showed strong immunoreactivity for the
-isoform in parietal cells and for the ß-isoform in pit cells of the stomach, along with the presence of these proteins in various other sites. Comparative staining revealed a similar but not identical distribution of I
B proteins in the Mongolian gerbil, a rodent model for H. pylori infection. The findings suggest that the
- and ß-isoforms are dominant I
B proteins in gastric parietal and foveolar cells, respectively, and point to a role for these transcription regulators in modulating pathological responses in stomach and other organs. (J Histochem Cytochem 48:191199, 2000)
Key Words:
transcription factors, IB, immunohistochemistry, gastric pit cells, gastric parietal cells
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Introduction |
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NF-B/I
B PROTEINS are major regulators of immune responses and inflammation (
B at steady state exists as a homo- or heterodimer in cytoplasm bound to I
B, which acts as a masking inhibitor. Many stimulants, such as tumor necrosis factor-
(TNF-
), interleukin-1 (IL-1), and lipopolysaccharides, activate the NF-
B/I
B complex. This activation entails phosphorylation of the I
B protein and its dissociation from NF-
B (
B translocates to the nucleus, where it acts as a transcriptional regulator (
NF-B proteins include p105 which, for example, associates at its C-terminal region with I
B
as the inhibitory component. Other members of the NF-
B family are associated with different inhibitory I
B isoforms. Members of the I
B protein family include I
B
, I
Bß, I
B
, I
B
, I
B
, and Bcl-3. Each of these isoforms possesses variable ankyrin repeats that connect the inhibitory protein to the Rel-homology domain of NF-
B protein. (
The NF-B/I
B system has been studied mainly in immunocompetent cells such as T- and B-lymphocytes and macrophages. However, NF-
B consisting of a p50/p65 heterodimer also has been demonstrated in cultured gastric surface epithelium (
B is upregulated in the stomach after H. pylori infection (
B/I
B complex in regulating gastric immune responses, because IL-8 expression is known to be regulated by NF-
B under certain conditions.
Although mRNA for IB
has been demonstrated by Northern blot analysis in gastric homogenates (
B protein isoforms have not been localized to specific cell types in the stomach. Moreover, there is little information concerning the distribution of members of the I
B family in other mammalian tissues. The present study undertook immunolocalization of I
B in stomach and in other organs for comparison. The results show a cell type-specific distribution of different I
B proteins in the gastric epithelium and in other tissues not previously known to express this transcription inhibitor.
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Materials and Methods |
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Animals and Tissue Processing
Tissues harvested from eight healthy 3-month-old mice of the BALB/c strain and four similarly aged Mongolian gerbils, Meriones unguiculatus (half from each gender), were examined. Care and use of the animals were approved by the Medical University of South Carolina's Animal Review Committee under NIH grant DC 00713. The animals were anesthetized by IP injection of sodium pentobarbital (50 mg/kg) and exsanguinated by transcardial perfusion with 10 ml of a 10% formalin solution containing 0.5% zinc dichromate, pH 5.0. The stomach and other organs, including eye, heart, lung, thymus, lymph nodes, liver, pancreas, small and large intestine, kidney, urinary bladder, ovary, uterus, testis, and seminal vesicles, were harvested, sliced into appropriately sized pieces, and immersed in the same fixative for 60 min. The specimens were dehydrated in a graded series of ethanols, cleared in Histoclear (National Diagnostics; Manville, NJ), and embedded in Paraplast X-TRA (Sherwood Medical; St Louis, MO) before sectioning at 5-µm thickness. Organs other than stomach were embedded together in composite blocks for appraising the tissue and cell type-specific distribution in the animal of IB proteins and the specificity of the antisera.
Immunohistochemistry
Immunostaining of tissue sections was performed as described previously (B
(C-21) 1:600; rabbit anti-mouse I
Bß (N-20) 1:300; rabbit anti-human I
B
(5177-C) 1:600; rabbit anti-mouse I
B
(M-364) 1:100 (Santa Cruz Biotechnology; Santa Cruz, CA). The sections were rinsed in PBS and incubated with biotinylated goat anti-rabbit IgG diluted 1:200. After rinsing with PBS, the sections were flooded with avidinbiotinhorseradish peroxidase complex (Vectastain ABC kit; Vector Laboratories, Burlingame, CA) for 30 min. The sections were again rinsed with PBS and reacted for 10 min in substrate medium containing 3,3'-diaminobenzidine HCl (Sigma Chemical; St Louis, MO) before dehydration and mounting.
Controls for immunostaining included the deletion of primary antiserum from the above-described staining procedure. In addition, each of the primary antisera against the -, ß-, and
-isoforms was preabsorbed with its homologous blocking peptide and with the other two available heterologous peptides to access crossreactivity. The
, ß, and
blocking peptides were purchased from Santa Cruz Laboratories. Blocking peptide for the
-isoform was not available.
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Results |
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Immunostaining demonstrated IB proteins in various histological sites in the mouse (Table 1) and gerbil. Specimens from mice and gerbils stained comparably, with the exception of a few minor differences noted below.
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Gastrointestinal Tract
Strong immunoreactivity for IB
was observed in cytoplasm of parietal cells in stomach (Figure 1a). The staining intensity varied slightly among individual cells, but not in accord with any known gradient. In contrast, I
Bß was localized in pit cells of the glandular epithelium (Figure 1b). In some deep foveolar cells the nuclei stained more intensely than did cytoplasm, whereas the most superficial cells exhibited pancellular reactivity (Figure 1b, inset). In the gerbil but not in the mouse, the peripheral cytoplasm and/or plasmalemma of parietal cells was weakly positive for I
Bß. The antiserum to I
B
stained superficial foveolar cells (Figure 1c), but the staining for I
B
differed from that for I
Bß in staining some but not other clusters of surface mucous cells.
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Cells positive for IBß and I
B
occurred sparsely in the corpus near the forestomach but were more abundant near the bottom of pyloric glands in mice (Figure 1d and Figure 1e). These cells corresponded with cells having clear, teardrop-shaped cytoplasm in H&E-stained sections (not shown), facilitating their designation as endocrine cells. In the gerbil these cells were positive for I
B
but not for I
Bß, and were scattered evenly in the lower region of the entire glandular epithelium.
Columnar cells in the upper half of villi in the mouse duodenum were reactive with antisera against IB
and I
Bß (Figure 2a and Figure 2b). Staining with anti-I
B
was stronger in nuclei than in cytosol in the positive cells. Moderate immunoreactivity for I
B
was detected in some cells at the apex of duodenal villi (Figure 2c). Although this pattern was similar in mice and gerbils, the gerbil tissues showed higher background staining and less intense nuclear staining for I
B
.
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Immune System
All four known IB isoforms were present in various regions of the spleen, thymus, and lymph nodes of both mouse and gerbil. Clusters of cells in splenic red pulp were strongly positive for I
B
(Figure 3a), whereas many isolated cells stained only weakly. Splenic cells expressed I
Bß and
similarly. I
B
immunostaining was confined to the marginal zone of white pulp in the spleen (Figure 3b).
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In the thymus, occasional cells with strong affinity for antibodies to IB
(Figure 3c), ß, and
lay scattered just under the capsule. Sporadic cells in the medulla of lymph nodes also showed strong immunoreactivity for I
B
, ß, and
(Table 1). These positive immune cells appeared more numerous in gerbil than in mouse.
Many IB-immunoreactive cells in immune system organs could not be clearly differentiated as lymphocytes, neutrophils, or macrophages. On the other hand, large reactive cells with abundant cytoplasm in pulmonary alveoli (Figure 3d), peritoneal fat around the ovary, and splenic red pulp were interpreted as macrophages. The presumed macrophages in these sites expressed both I
B
and ß.
Genitourinary System
Cytoplasm in epithelium of the murine seminal vesicle showed moderate to intense staining with anti-IB
(Figure 4a) and weaker staining with anti-I
B
. In the mouse testis, the heads of spermatids showed moderate reactivity, and a small number of presumably meiotic spermatocytes were intensely stained with anti-I
Bß (Figure 4b and inset). In the gerbil, spermatid/spermatozoon reactivity was abundant, whereas spermatocyte staining was rare.
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Cytosol in epithelium of the endometrium (Figure 4c) and oviduct (Figure 4d) stained moderately for the IBß-isoform. Cytosol of oocytes revealed moderate staining for both I
B
(Figure 4e) and I
B
. In urinary bladder, basal cells of the transitional epithelium expressed strong immunoreactivity demonstrative of I
Bß (Figure 4f).
Miscellaneous Organs
In the lung, some bronchiolar cells showed moderate to strong cytosolic affinity for IB
(Figure 5a) and I
Bß antibodies, as did occasional cells infiltrating pulmonary alveoli (Figure 5b). Most of the latter cells possessed a polymorphic nucleus and were considered to be neutrophils, but a few with abundant cytoplasm and an oval nucleus were interpreted as alveolar macrophages. Pneumocytes lacked staining.
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IB
and I
B
were restricted to cells presumably representing
- and/or
-cells in the periphery of pancreatic islets (Figure 5c). Cytoplasm of the positive islet cells was stained intensely. The centrally located ß-cells bound antibody to
- and
-isoforms only weakly.
In palpebral conjunctiva, the most superficial cells exhibited intense staining for IBß, far exceeding that in the basal palpebral conjunctiva superficial corneal epithelium (Figure 5d).
Deletion of the primary antiserum resulted in no staining in any tissue. Absorption of either anti-IB
or anti-I
Bß with its homologous blocking peptide completely eliminated staining in all sites. In contrast, I
B
blocking peptide diminished but failed to abolish lymphocyte immunoreactivity with anti-I
B
in the spleen. Cross-absorption of the primary antisera with similar concentrations of the heterologous blocking peptides (
, ß, and
) failed to affect staining reactivity in any site.
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Discussion |
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Cellular Distribution of IB Proteins
Although NF-B proteins were first detected in the immune system, they are now known to exist in other organs such as brain (
B system has also been observed in fibrocytes (
B/I
B proteins.
This report presents the first immunohistochemical data mapping the expression of inhibitor IB isoforms in several major organ systems. The study shows a widespread distribution of immunodetectable NF-
B/I
B proteins. Previous in situ hybridization data (
B
distributed diffusely throughout the spleen appear not to agree with the present observation of focal strong staining for this isoform in red pulp and only weak reactivity in the marginal zone and lymphatic follicles. The reason for a discrepancy between the in situ hybridization and immunohistochemistry results is uncertain, but a similar observation has been made for RelA(p65) and NF-
B1(p50) (
IB
has been detected in Western blots prepared from a bladder cancer cell line (
Bß immunostaining observed here only in basal cells of the transitional epithelium.
Prior evidence existed for activity of the NF-B/I
B system in pancreatic ß-cells based on production of nitric oxide synthase after NF-
B activation by IL-1ß stimulation (
B
and
at high concentration only in peripheral islet cells, in contrast to a much lower staining intensity in central ß-cells.
Conjunctival and endometrial adenocarcinoma cell lines possess NF-B protein (
B/I
B complex in conjunctival and endometrial epithelia. Our present results concur with this observation and identify the inhibitory component in both cell types as I
Bß.
The perception of the NF-B/I
B system as a complex that dissociates on activation, releasing NF-
B protein into the nucleus (
B proteins in the cytoplasm in some cells but in the nucleus or in both locations in others. Duodenal epithelium, mitotic spermatocytes, and deep gastric foveolar cells, for example, disclosed nuclear staining for I
B
- or ß-isoforms. Although the nuclear distribution of I
B proteins is not fully understood, some recent work indicates that nuclear staining reveals newly synthesized I
Bß (
B in the nucleus induces synthesis in cytoplasm of I
Bß protein, which, lacking phosphorylation, binds to but fails to inhibit NF-
B. Translocation of this active NF-
B/I
B complex to the nucleus, where it prolongs transcriptional activity, could explain the nuclear immunostaining for I
Bß. On the other hand, newly produced I
B
migrates into the nucleus and transports NF-
B to the cytoplasm. This NF-
B/I
B
complex, which has the effect of decreasing NF-
B activity in the nucleus (
B
.
NF-B/I
B Proteins in Stomach
NF-B has been detected in gastric mucous cells and parietal cells (
B isoform(s) that inhibits NF-
B proteins in gastric mucosa has not been determined. Our present study provides further evidence for the NF-
B/I
B system in the gastric mucosa and identifies I
Bß as the major inhibitory isoform in the pit cells.
One proposed activity of the NF-B/I
B system in pit cells concerns induction of IL-8, which is a chemotactic cytokine. H. pylori infection increases IL-8 immunoreactivity in pit cells (
B, because H. pylori infection has been shown to activate NF-
B in gastric biopsy specimens (
B activation (
B in pit cells results from the dissociation of the I
Bß isoform. That the I
B
and I
Bß isoforms regulate NF-
B activity differently is suggested by their different kinetics (
IB protein has not been demonstrated previously in gastric parietal cells, although mRNA for I
B
is present in the stomach (
B
in parietal cells points to a role for transcriptional regulation in this cell type through dissociation of I
B
from NF-
B. Phosphorylation of I
B proteins mediates this separation. Information concerning the phosphokinases that phosphorylate I
B proteins is limited. It is known that casein kinase II phosphorylation of the C-terminus of I
B
and ß occurs unrelated to inducible activation of NF-
B, whereas phosphorylation of the N-terminus of these isoforms by I
B kinases (IKKs) induces dissociation of I
B (
B protein in parietal cells. Gastric parietal cells are known to contain several phosphokinases, but whether these phosphorylate I
B
has not been determined.
A function proposed for the NF-B/I
B system in parietal cells is the induction of prostaglandin endoperoxide synthase-2 (COX-2). H. pylori infection induces COX-2 expression in gastric parietal and pit cells (
B have been shown to induce COX-2 in several different cell types (
B
may play a role in upregulating COX-2 in these systems and, by analogy, also in gastric parietal cells. Because H. pylori infection activates NF-
B, it is likely that this system mediates the increase of COX-2 in parietal cells of H. pylori-infected stomach and that the I
B
observed in these cells functions in this response.
Absorption of antiserum with homologous peptide abolished affinity for antibodies to IB
and ß, whereas absorption with the heterologous peptide did not, establishing specificity of the staining for these two I
B isoforms. The lack of I
B
-specific peptide precluded absorption studies for this isoform. Although data reported here offer the first indication of the presence of I
B
in stomach, the specificity of staining for this isoform remains inconclusive because of failure of blocking peptide to completely inhibit staining with anti-I
B.
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Acknowledgments |
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Supported by Research Grant DC 00713 from the National Institute on Deafness and Other Communication Disorders, National Institutes of Health.
We thank Ms Deborah Brown for editorial and Ms Barbara Schmiedt for technical assistance.
Received for publication September 10, 1999; accepted September 15, 1999.
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