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Original Studies |
Division of Cell Biology and Experimental Cancer Research, Institute of Pathology, University of Berne (J.C.R., J.A.L., B.W.), Berne, Switzerland; the Department of Cell Biology, Baylor College of Medicine and Biomedical Computing, Inc. (D.L.S.), Houston, Texas 77030; and the Department of Integrative Biology, Pharmacology, and Physiology, University of Texas Houston Medical School (R.W.H., A.S.), Houston, Texas 77030
Address all correspondence and requests for reprints to: Jean Claude Reubi, M.D., Division of Cell Biology and Experimental Cancer Research, Institute of Pathology, University of Berne, P.O. Box 62, Murtenstrasse 31, CH-3010 Berne, Switzerland. E-mail: reubi{at}patho.unibe.ch
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Abstract |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Five subtypes of somatostatin receptors, designated sst1 to sst5, have recently been identified (9). Investigations in rats and humans using RT-PCR and in situ hybridization have shown that the established somatostatin targets do express somatostatin receptor messenger ribonucleic acid (mRNA), often for several receptor subtypes concomitantly (10, 11, 12). One of the subtypes frequently identified by mRNA studies is the sst2 subtype. In human and rodent tissues the sst2 receptor has been shown to exist in two isoforms, sst2A and sst2B, generated through alternative splicing of the sst2 mRNA at the 3'-end of the coding segment (13, 14). These two variants, which differ only in length and in their amino acid sequence at the C-terminals, exhibit similar binding properties but vary in G protein coupling (13).
Recently, a specific polyclonal antibody (R2–88) was generated against a unique sequence, CERSDSKQDKSRLNETTETQRT, located at the C-terminal tail of the sst2A receptor (15). With this tool, the sst2A receptor protein could be identified with immunohistochemistry in the rat brain, pituitary, and pancreas (15, 16, 17) as well as in human tissues, including cancers and pancreas (18, 19). Not only were the proteins for a specific somatostatin receptor identified, but a better cellular resolution could be achieved with this method.
Despite receptor autoradiographical evidence showing that somatostatin receptors are expressed and localized in discrete regions of the human lymphoid system and peripheral nervous system (5, 20), the exact anatomical site and subtype of the receptor proteins have not been identified in these tissues to date. The aim of the present study was, therefore, to evaluate normal somatostatin targets such as the peripheral nervous system, the lymphoid system, and the smooth muscles for their content in sst2A protein with immunohistochemical methods using R2–88 antiserum. The human intestinal tract was chosen as an important source of peripheral nervous system tissue in the form of the submucosal and myenteric plexus, of lymphoid tissue such as in Peyer’s patches and solitary lymphoid follicles, and of smooth muscle with its circular and longitudinal coat. R2–88 immunohistochemistry was compared to receptor autoradiography and/or with Western blots in these tissues.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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. The
samples were either frozen or formalin fixed immediately after
resection.
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The R2–88 rabbit polyclonal antibody, generated against a unique sequence located in the C-terminal tail of the sst2A receptor was used as primary antibody (15). Biochemical and immunohistochemical characterization of the antibody have been reported previously (15, 21). R2–88 immunohistochemistry was essentially performed as described previously (18).
Frozen tissues. Ten-micron thick sections were cut on a cryostat (Leitz, Rockleigh, NJ). The sections were fixed for 10 min in acetone and 10 min postfixed in 4% paraformaldehyde (diluted in phosphate-buffered saline), and incubated for 20 min in 5% normal goat serum diluted in Tris-buffered saline (TBS). Then, the sections were incubated with the R2–88 antibody against the sst2A receptor overnight at room temperature. The antibody R2–88 was used at a 1:6000 dilution in TBS containing 1% BSA, 5% normal goat serum, and 0.1% NaN3. Sections were incubated for 45 min at room temperature in a 1:200 dilution (same buffer as for primary antibody) of biotinylated goat antirabbit Ig antiserum (DAKO Corp., Glostrup, Denmark) and thereafter for 45 min at room temperature with avidin-biotin complex/horseradish peroxidase (1:120 in TBS; DAKO Corp.). Finally, sections were developed in 0.05% 3,3'-diaminobenzidine (Fluka) and 0.006% H2O2 (Merck & Co., Inc., Rahway, NJ), weakly counterstained with hematoxylin, and mounted. A tissue was considered positive for R2–88 when the immunostaining was abolished after absorption of the antibody with the peptide antigen at 100 nmol/L (30 min at room temperature, with agitation before application of the antibody to the tissue). The tissue reaction was considered negative if the immunostaining was not suppressed in the presence of the antigen.
Formalin-fixed, paraffin-embedded tissues. In all samples tested, the fixation time was 24–36 h. The fixed tissue was processed for conventional, 2- to 5-µm thick paraffin wax (Paraplast) sections. The sections were dewaxed, rehydrated, and boiled in 10 mmol/L citrate buffer, pH 6.0, in a pressure cooker as described previously (18). Sections were then (and after all subsequent steps) washed in TBS and incubated with the R2–88 polyclonal antibody against sst2A receptors used at a dilution of 1:2000 overnight at room temperature. All subsequent steps, including absorption of the antibody with the peptide antigen, were performed exactly as in the protocol for frozen tissue, and the same criteria were applied to distinguish between positive and negative tissues.
Other immunohistochemical markers
The immunohistochemical staining of synaptophysin for optimal identification of the submucosal and myenteric plexus was performed on cryostat and formalin-fixed material, using commercially available antibodies (DAKO Corp.) at a dilution of 1:100.
Receptor autoradiography with 125I-[Tyr3]octreotide
Twenty-micron cryostat sections, adjacent to those used for R2–88 immunohistochemistry, were used for in vitro receptor autoradiography with the sst2-preferring radioligand, 125I-[Tyr3]octreotide, as described previously (22). Nonspecific binding was determined in the presence of 10-6 mol/L octreotide.
Immunoblots
Cell membranes from the sst2A-expressing rat GH-producing pituitary cell line GH-R2 cells were prepared as previously described (23). Human smooth muscle tissue was dissected from the circular and longitudinal muscles of the human colon using magnifying glasses, and membranes were prepared following previously described procedures (18). Membrane proteins were solubilized in sample buffer [62.5 mmol/L Tris-HCl, 2% SDS, 10% 2-mercaptoethanol (vol/vol), 6 mol/L urea, and 20% glycerol, pH 6.8], separated by SDS-PAGE on a 7.5% polyacrylamide gel, transferred to a polyvinylidene difluoride membrane, and immunoblotted with R2 88 antiserum as described previously (23).
Peptide sequence analysis
The BLAST server at NCBI (http://www.ncbi.nlm.nih.gov/BLAST/) (24, 25) was used to search the nr database (All non-redundant GenBank CDS translations + PDB + SwissProt + PIR + PRF). Several different methods for performing the search were used. Both the Old (ungapped) BLAST program and the BLAST 2 (Gapped BLAST) program were used. The following amino acid substitution scoring matrixes were used: BLOSUM45, BLOSUM62, BLOSUM80, PAM30, and PAM80 for BLAST 2, and BLOSUM62 and PAM40 for Old BLAST. In every case the EXPEC parameter was increased from the default of 10 to a value of at least 1000, and in one case up to 5 x 108. All of these parameters are explained in http://www.ncbi.nlm.nih.gov/BLAST/. The remaining software used for these analyses was supplied as parts of the GCG package, version 9.1, installed at Academic Informatics Services, Baylor College of Medicine (Houston, TX).
A set of 14 full-length or near full-length mammalian myosin heavy chain sequences were identified by searching Entrez (http://www.ncbi.nlm.nih.gov/Entrez/) for the keywords myosin, heavy, and complete. The resulting matches were visually examined, and relevant sequences were downloaded. The sequences used were Entrez Protein Database accession no. 219524, 3041706, 547966, 189034, 3043372, 3041707, 88201, 547981, 1945078, 1945080, 109322, 1346644, 165490, and 127755.
These 14 sequences and the 21-residue sst2A antigen peptide sequence were aligned with the Pileup program of GCG, using default parameters. The Distances program was used to determine the mutational distance between these sequences. Finally, the Bestfit program was used to find the best pairwise alignment of the immunizing peptide and one representative of skeletal and smooth muscle myosin sequences: PIDs 3041707 and 1945078, respectively.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Germinal centers of solitary lymphoid follicles in the colon, of
Peyer’s patches in the ileum, and of lymphoid follicles in the
appendix were labeled with the sst2-preferring
125I-[Tyr3]octreotide radioligand in receptor
autoradiographic studies (Table 1
). Figure 1 is an example of a receptor
autoradiography experiment showing somatostatin receptor-positive
germinal centers in a Peyer’s patch labeled with
125-[Tyr3]octreotide. Such germinal centers
could also be immunostained specifically with the R2–88 antibody. The
immunostaining was usually weak to moderate and was found in the
germinal center forming the central part of the follicle. Figure 2 shows an example of an
R2–88-immunoreactive germinal center in a Peyer’s patch. The luminal
part of the germinal center was more heavily stained than the basal
part, confirming the observation of polar labeling of germinal centers
made previously with receptor autoradiography (20), reflecting the
normal polarity of germinal centers (26). As a specificity control,
antibody preabsorption with peptide prevented immunostaining of the
germinal center completely (Fig. 2). At high magnification the R2–88
immunostaining revealed features not detected by autoradiography,
namely a preferentially membranous localization of the sst2A receptors
(Fig. 2). This membrane staining was best seen when the sections were
not counterstained with hematoxylin-eosin.
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The submucosal and myenteric plexus of the human colon could be
clearly visualized with receptor autoradiography using
125I-[Tyr3]octreotide as radioligand. Figure 3 shows a typical example of the
myenteric plexus, identified by synaptophysin immunoreactivity, that is
strongly radiolabeled with
125I-[Tyr3]octreotide. These neural plexuses
could also be clearly immunostained with R2–88. Figure 4 shows an example of a myenteric plexus
characterized by its strong synaptophysin immunoreactivity; the plexus
was strongly immunoreactive for R2–88; immunoreactivity was primarily
found over nerve elements entering and leaving the plexus and localized
around the ganglion cells. In addition, the immunostaining was
compatible with sst2A receptors localized on the cell membranes of the
ganglion cells. Antibody preabsorption with 100 nmol/L of the antigen
peptide completely prevented the staining. Figure 4 shows also an
example of an R2–88-immunoreactive submucosal plexus of the colon,
with negative control of antibody preabsorption using 100 nmol/L
peptide antigen. The tested plexuses were immunoreactive for R2–88 in
both cryostat material as well as formalin-fixed, paraffin-embedded
material. As an illustration for the latter, Fig. 5 shows the discrete R2–88
immunoreactivity seen in the nerve fibers of a myenteric plexus from a
formalin-fixed, paraffin-embedded 3-µm thick section.
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Somatostatin receptor autoradiography did not detect significant
amounts of somatostatin receptors in gastrointestinal colonic smooth
muscle, in either the circular or the longitudinal coat. Nonetheless,
R2–88 immunostaining was observed in all smooth muscle tissues in the
colon, including the circular and longitudinal coats (Fig. 4) and the
smooth muscle of large vessels. Moreover, antibody preabsorption with
100 nmol/L peptide antigen substantially inhibited the immunostaining,
indicating that staining was produced by receptor peptide antibody. To
further evaluate the specificity of the immunoreactivity in muscle
tissue, Western blots were performed on carefully dissected pieces of
colonic smooth muscle. The results shown in Fig. 6 demonstrate the characteristic staining
of a broad 80-kDa band corresponding to the sst2A receptor in a
pituitary cell line transfected with a clone encoding this receptor
subtype (18, 21). In contrast, only a 206-kDa band was stained in
smooth muscle membranes. This high molecular mass band comigrated with
rabbit skeletal muscle myosin, the 206-kDa protein standard.
Interestingly, rabbit myosin was weakly stained by the R2–88
antiserum. These results suggest that the R2–88 antiserum cross-reacts
with myosin and that this cross-reactivity is responsible for the
immunocytochemical staining observed in human colonic muscle.
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An important consideration in the choice of the particular peptide
used to raise the R2–88 antibody was that a BLAST search of GenBank
retrieved the known somatostatin receptor type 2 proteins but no
others, thus making it a unique sequence (21). However, a perfect match
is not required for antibody cross-reactivity. In response to the
cross-reactivity of the R2–88 antiserum with myosin heavy chain
observed in Fig. 6, we conducted a series of analyses to determine
whether this cross-reactivity could be explained by partial sequence
similarity between the peptide and myosin. We first adjusted the BLAST
parameters to increase the sensitivity of the search. Of all the
parameters we used to search GenBank (see Materials and
Methods), use of the PAM30 matrix provided the most sensitive
search. Using an EXPECT value of 1000, instead of the default value of
10, we retrieved 181 sequences: 6 known somatostatin receptor 2
sequences and 175 other sequences. None of these sequences was myosin
heavy chain sequences. Ultimately, by increasing the EXPECT value to
5 x 108, we retrieved 2918 sequences. Although among
these were 60 myosin sequences, only 9 of these were the expected
mammalian smooth or skeletal muscle myosins. We conclude that these
matches are of questionable significance and could not reasonably have
been used to predict the myosin cross-reactivity.
To determine whether the region of sequence responsible for cross-reactivity could be identified, we analyzed a collection of 14 mammalian smooth and skeletal muscle myosin heavy chain sequences. We constructed a multiple alignment of these sequences to identify the homologous regions. As has been previously reported (27), the myosin sequences fell into 2 groups, with all of the smooth muscle myosins in 1 group, and the skeletal and other myosins falling into 2 related groups. There was only a slight degree of sequence similarity between the smooth and skeletal muscle myosins. Finally, we performed a rigorous pairwise alignment between the peptide sequence and a representative of the smooth and skeletal groups. The Bestfit algorithm used can almost always detect an alignment and, as expected, pairwise alignments were detected in this case. Two points about these alignments are significant, however. First, the optimal alignment between the peptide sequence and the different myosin sequences did not occur at homologous sites, but, rather, at different sites in the sequence, suggesting that these alignments were not significant. Second, a random match control was included in these analyses, and in all cases this control indicated that the optimal alignments with the antigen peptide were not of significantly higher quality than that expected by chance. Taken together, these results demonstrate that standard sequence alignment software cannot identify the sequences in myosin responsible for the observed reactivity with the R2–88 antiserum. This should not be taken as evidence that the observed reactivity is not directed toward a specific sequence in skeletal and smooth muscle myosins, however. The observed reactivity is presumably directed against a primary sequence feature, as it is detected by Western blotting, but the scoring matrexes used in these analyses have been optimized for detecting evolutionary relationships rather than features important for antibody recognition and thus could well have missed the relevant sequence.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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As somatostatin-immunoreactive neurons have been shown to be present in the myenteric plexus (29), it is likely that the sst2A receptors in the myenteric plexus may be mediating neuronal functions. They may be involved in the regulation of peristalsis and interdigestive motility. Which of the various neurons present in the plexus (sensory neurons, interneurons, inhibitory and excitatory motor, and secretomotor neurons) are exactly involved in the somatostatin action (30) cannot be precisely evaluated in the present study. The presence of sst2A receptors in submucosal plexus may be related to a function of somatostatin in regulating absorptive and secretory mechanisms of the mucosa as well as regulating blood flow (29).
There are two potential local sources of endogenous somatostatin that could interact with sst2A receptors expressed by the germinal centers of the lymphoid follicles. First, somatostatin originating from nerve fibers of the peripheral vegetative nervous system, which have been shown to reach lymphatic follicles of the gastrointestinal tract in animals (31). Second, somatostatin produced by immune cells of lymphoid organs (32, 33). In such cases, sst2A receptors may mediate the neural and/or immune regulation by somatostatin of humoral antibody responses, as discussed in more detail previously (34).
The strong immunoreactivity found in the gastrointestinal mucosa was shown to be due to nonspecific staining by an unrelated antibody in the R2–88 serum, as preincubation of the serum with antigen peptide did not inhibit this staining. Unfortunately, this nonspecific staining may mask weak specific staining in this tissue, and hence, low abundance sst2A receptors cannot be detected. In contrast, the homogeneous immunostaining of all smooth muscles of the gastrointestinal tract could be inhibited by antibody absorption with 100 nmol/L peptide antigen, indicating that it was due to antipeptide antibody. Nonetheless, this smooth muscle staining is unlikely to result from sst2A receptors, because 1) receptor autoradiography using sst2A-preferring radioligand was negative in all circular and longitudinal smooth muscles; and 2) Western blots of dissected circular or longitudinal gastrointestinal smooth muscle did not show a characteristic 80-kDa sst2A band; rather, a 206-kDa band, comigrating with rabbit myosin heavy chain was stained. Furthermore, pure rabbit skeletal muscle myosin heavy chain, used as a protein standard, was also weakly stained by the R2–88 antiserum. Knowing the amounts of rabbit myosin heavy chain and sst2A receptor loaded on the gel and estimating the relative intensity of the R2–88 staining indicated that the R2–88 antiserum detected the sst2A receptor with approximately 60,000 times the sensitivity that it detected myosin.
Three observations indicate that human myosin is likely to be the cross-reacting protein in smooth muscle: 1) the sst2A receptor peptide antibody exhibits weak cross-reactivity with rabbit myosin; 2) the antibody recognizes a protein in smooth muscle that comigrates with rabbit myosin heavy chain; and 3) myosin is known to be present in high concentrations in smooth muscle. If this conclusion is correct, it is important to note that the cross-reactivity is detectable only in the presence of a vast excess of the cross-reacting protein. Thus, it is noteworthy that the R2–88 antiserum does not detect a 206-kDa protein in brain, pituitary, or pancreas (15, 16, 17). Interestingly, recent studies have shown that antibodies found in sera of myasthenia gravis patients against another neurotransmitter receptor, namely the acetylcholine receptor, can also cross-react with muscle myosin (35). As muscle tissues have high levels of myosin, such a cross-reactivity, even when weak, is of general concern.
Although circular and longitudinal smooth muscles of the human gastrointestinal tract have not been found in the present study to express measurable amounts of sst2A receptors, it does not mean that all human smooth muscles are lacking such receptors. We have indeed reported previously that smooth muscles of human veins located in the immediate surroundings of various tumors (36) or in inflammatory bowel diseases (6) can express somatostatin receptors; they probably belong to the sst2 type, as they are readily identified with 125I-[Tyr3]octreotide autoradiography. Also the smooth muscles surrounding the normal human prostate glands express a low to moderate density of such somatostatin receptors (37). For comparison, in the rat gastrointestinal tract, Krempels et al. (11) reported the presence of mRNA for sst2 in the smooth muscle layers and in the blood vessels. Unfortunately, the cross-reactivity of R2–88 with myosin will make it difficult to identify sst2A receptors with this antibody in human smooth muscle tissues, including vascular and prostatic smooth muscles as well as gastrointestinal smooth muscle layers, due to a probable masking of the specific sst2A staining by the myosin staining.
One common argument used to support the specificity of antibodies used for immunocytochemistry is that the peptide antigen against which the antibody was raised has a unique sequence. This argument together with the observation that immunoreactivity can be blocked by excess peptide are often offered as proof of the identity of the reacting protein. However, we show here that even using the most extreme criteria, the sst2A antigen peptide has no significant similarity to protein sequences in the databanks in general or to the putative cross-reacting myosin sequences in particular. We suspect that the antibody does cross-react with a specific common peptide sequence within these myosin proteins, but that the current sequence alignment tools are unable to identify this region. However, the important conclusion of our analysis is that the inability to detect similar sequences in the protein databases is no guarantee that antibodies raised against a peptide will not cross-react with other cellular proteins. Specific functional and biochemical tests of specificity, such as the ligand binding and molecular mass characterization in immunoblots as reported here, are necessary to confirm the specificity of any novel antibody used in immunohistochemical investigation.
In summary, we have shown that the sst2A receptor protein is present in the peripheral nervous system and in the lymphatic system of the human gastrointestinal tract. These observations may be clinically relevant; indeed, the sst2-preferring drug octreotide, widely used in various somatostatin receptor-expressing endocrine and gastrointestinal pathologies, is also likely to specifically act during treatment of these diseases on other sst2A targets, such as the lymphoid system and the peripheral nervous system. The search for the precise octreotide action through these sst2A targets, which may either be beneficial in certain pathologies or may lead to undesirable side-effects, will be of considerable interest.
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Footnotes |
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Received February 2, 1999.
Revised April 14, 1999.
Accepted April 16, 1999.
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