More than 15 subunits of the integrin family of cell surface adhesion molecules have been identified. The α6β4 integrin has recently been identified as a component of hemidesmosomes of stratified squamous epithelium. The monoclonal antibody (mAb) 346-11A binds to the β4 subunit in mice. Sequence analysis of a cDNA clone coding for this epitope localizes reaction of the mAh to a portion about half way through the extracellular domain at the beginning of the cysteine-rich region.

Sites of β4 expression in mice were detected by autoradiographic analysis of tissues collected from mice 24 or 48 h after intravenous injection of 125I-labeled mAh 346-11A. This non-quantitative technique emphasizes detection of antigen exposed in the vascular space.

These data show that in addition to the epithelia of several organs, the endothelia of intermediate vessels throughout the body are sites of β4 expression. In particular, endothelium of larger vessels but not capillaries in lung, vessels in thymus, spleen and Peyer’s patches, and portal vessels but not the central veins of the liver, are positive. The expression of the fβ4 integrin in blood vessels may indicate a specialized function for β4 at these sites that is distinct from its role in hemidesmo-some-mediated attachment.

The integrins are an ever-expanding family of α, β heterodimers that function in cell adhesion (Hynes, 1987; Ruoslahti and Pierschbacher, 1987; Hemler, 1988; Buck and Horwitz, 1987; Humphries, 1990; Ruoslahti, 1991). While specific ligands are known for certain of the integrins, their role in establishing and preserving organ architecture is not clear. Some integrins are widely distributed (De Strooper et al., 1990) and detailed studies on cell lines of various origins show expression of several different integrins.

The focus of this work is the distribution of the β4 subunit in vivo. To date, the β4 subunit has been found invariably associated with the α6 subunit (Kajiji et al., 1987; Kennel et al., 1986; Kennel et al., 1989; Sonnenberg et al., 1990a). The subunit can complex with the β1 subunit (termed VLA-6) (Hemler, 1988) to form a laminin receptor (Sonnenberg et al., 1990b; Sonnenberg et al., 1988). Distribution of the α6β1 laminin receptor is extremely broad including expression on platelets, lymphocytes, macrophages and nearly all epithelial tissue (Sonnenberg et al., 1990a).

One hint of the function of a particular integrin is the site and level of its expression in vivo. Several such studies have been done by immunoperoxidase or immunofluorescence analyses of tissue sections, which show that the β4 subunit is much more restricted than in its expression (Kajiji et al., 1987; Sonnenberg et al., 1990a). Several groups have established its presence on organ epithelium and on many carcinomas (Tamura et al., 1990; Kennel et al., 1989; Stepp et al., 1990; Carter et al., 1990; Kajiji et al., 1987; Sonnenberg et al., 1990a). Immunohistochemistry on normal tissue has demonstrated that β4 co-localizes with Bullous Pemphigoid Antigen (BPA) in hemidesmosomes of skin (Carter et al., 1990; Sonnenberg et al., 1990c) and cornea (Stepp et al., 1990). Recent work from our laboratory has shown that mAb injected intravenously (i.v.) localizes very efficiently at sites inside the vascular space (Kennel et al., 1990; Kennel et al., 1991). We reasoned that if β4 integrin were expressed at intervascular sites, it might be more easily detected by this method.

In this paper, we show that, in addition to epithelial sites, β4 subunit is expressed on the endothelium of medium-sized blood vessels, but not capillaries, of several organs in the mouse.

The origin and specificity of rat mAb 135–13C (Kennel et al., 1981) to the α6 subunit of integrin (Sonnenberg et al., 1990a; Kennel et al., 1989) and of mAb 346–11A (Kennel et al., 1986) to the β4 subunit have been previously described (Kennel et al., 1989). The murine lung alveolar type II cell carcinoma, Line 1 (Yuhas et al., 1975), was grown in McCoy’s 5A medium supplemented with glutamine, penicillin and streptomycin as described (Kennel et al., 1981).1

125I-labeled mAb distribution

Purified mAb 346–11A or control mAb 135–14 were radioiodinated with chloramine T to a specific activity of about 10,000 counts/min per ng (see figure legends for exact specific activity) and repurified as described previously (Kennel et al., 1990). Labeled mAb was injected i.v. in 100 μl 10 mM sodium phosphate, pH 7.6, 0.15 M NaCl (PBS) containing 5 mg/ml bovine serum albumin into 12-week-old Balb/c Bd female mice from the Oak Ridge National Laboratory colony. Animals were killed at 24 h or 48 h after injection, the tissues were fixed in buffered formalin and embedded in paraffin. Sections (5 μm) were cut and processed for autoradiography (Kennel et al., 1990). Slides were exposed to emulsion 10 days before developing and staining with hematoxylin and eosin.

cDNA cloning

A ‘stretched’ cDNA library from poly(A)+ containing Line 1 RNA (see below) was prepared in λgtll by Clontech Labs (Palo Alto, CA). The library was screened on Escherichia coli Y1090 (Young and Davis, 1983) with a rabbit polyclonal antibody to purified murine α6β4 complex. Positive phage plaques were picked and amplified, and DNA was prepared by the plate lysate method (Sambrook et al., 1989). DNA was amplified from the λgtll recombinant template with the polymerase chain reaction (PCR) using commercial λgtll primers (New England Biolabs, Beverly, MA) and a GenAmp™ kit (Perkin-Elmer Cetus, Norwalk, CT). A Perkin Elmer Cetus thermal cycler was used according to the following program. Cycle 1:94°C, 3 min; 40°C, 1 min 30 s; 72°C, 2 min. The first cycle was followed by 34 cycles: 94°C, 1 min; 40°C, 1 min, 72°C, 1 min 30 s. These 34 cycles were followed by a final 7 min 72°C extension and shutdown cycle.

Amplified DNA was analyzed on agarose gel electrophoresis and subcloned into the EcoRI site of pGEM-7Z (Promega, Madison, WI). DNA prepared from clones containing the inserted cDNA were sequenced with a Sequenase 2.0 sequencing kit (United States Biochemical Corp., Cleveland, OH) according to the manufacturer’s directions. Sequences from two pGEM-7Z clones from independent PCR amplifications were found to be identical.

Northern blot analyses

Total RNA from Line 1 cells grown in 100 mm dishes was isolated by a modification of the procedure of Birnboim (1988). Isolated total RNA (Kingston, 1989) or poly(A)+-containing RNA (Sambrook et al., 1989) was separated on 1% agarose gels in 10 mM sodium phosphate buffer, pH 6.5, containing 2.2 M formaldehyde (Lehrach et al., 1977). The gel was rinsed with 20 × SSC (1 × SSC is 15 mM sodium citrate, pH 7.0, 0.15 M NaCl) and transferred to a Genescreen™ (New England Nuclear Products, Boston, MA) nylon support overnight in 20 × SSC. The nylon blot was UV irradiated (258 nm) at 1200μW/cm-2 for 2 min before wetting in 2 M NaCl, 10 mM Tris-HCl, pH 8.0, 0.1% SDS at 45°C for 20 min. Hybridization was carried out in 8 ml of 7% SDS, 0.5M sodium phosphate buffer, pH 7.2, 2 mM EDTAand 1% nonfat dry milk at 60°C for 18 h (Church and Gilbert, 1984). Probes were 10 ng of acrylamide gel-purified (Sambrook et al., 1989) DNA labeled by random primer DNA synthesis with an oligolabeling kit (Pharmacia, Uppsula, Sweden). Probes were PCR DNA of 16 Ab 34A clone, 355 bases and a BgμI, Avail fragment of 250 bases from a human cDNA y-actin clone (Gunning et al., 1983). After probing, blots were washed twice in 2x SSC and 0.1% SDS for 30 min at 60°C and then 3 times in 0.2× SSC and 0.1% SDS for 30 min each time.

β4sequence containing the 346-11A epitope

Line 1 lung carcinoma was used to prepare a cDNA library in λgtll. The library was screened with rabbit polyclonal antibody to the purified α6β4 complex. Of 17 positive clones, one (16Ab34) was found to be positive for reaction with mAb 346–11A, the prototype mAb to murine β4 (Kennel et al., 1989; Sonnenberg et al., 1990a). Western blot analyses of isopropyl-β-D-thio-galactoside (IPTG)-induced proteins from clone 16Ab34 showed a fusion protein of about 130 kDa detected with mAb 346–11A (data not shown). This cDNA of 355 bases is highly homologous to the human β4 sequence (Fig. 1) and has an open reading frame with 103 of 117 residues in common with human β4 amino acid sequence, although this mAb does not bind to the human)34 subunit (data not shown). This region of the protein containing the 346–11A epitope starts at amino acid 433 and is at the beginning of the Cys-rich region. The human μ4 and ft proteins share only 49 of 117 amino acids in this stretch and 17 of these common residues are Cys residues that are conserved in all β subunits (Hogervorst et al., 1990; Suzuki and Naitoh, 1990).

Fig. 1.

Nucleotide sequence of murine lung tumor cDNA from clone 16Ab34 reactive with mAb 346–11A. The sequence is compared with the human sequence by the numbering system of Hogervorst et al. (1990). Identical bases are designated by a dot. The human deduced protein sequence has been divided into regions: signal peptide (▪); extracellular domain (▪); Cys-rich region (▪); transmembrane domain (▫); and cytoplasmic domain (▫). These sequence data are available from EMBL/GenBank/DDBJ under accession number ×58254.

Fig. 1.

Nucleotide sequence of murine lung tumor cDNA from clone 16Ab34 reactive with mAb 346–11A. The sequence is compared with the human sequence by the numbering system of Hogervorst et al. (1990). Identical bases are designated by a dot. The human deduced protein sequence has been divided into regions: signal peptide (▪); extracellular domain (▪); Cys-rich region (▪); transmembrane domain (▫); and cytoplasmic domain (▫). These sequence data are available from EMBL/GenBank/DDBJ under accession number ×58254.

Insert cDNA purified from clone 16Ab34 was used to probe RNA samples for mRNA expression (Fig. 2). A 5.9 kb message was detected in total RNA samples from Line 1 cells (lane 5) and in mRNA from thymus (lane 1), uterus (lane 2), skin (lane 3), and Line 1 cells (lane 4). No other significant size forms of β4 mRNA were detected with this probe or with a probe from the cytoplasmic domain (data not shown).

Fig. 2.

Northern blot analyses of RNA samples from various murine cells and tissues. Poly(A)+-containing RNA from thymus, 5 μg (lane 1); uterus, 5 μg (lane 2); skin, 5 μg (lane 3) or Line 1 cells, 5 μg (lane 4); or total RNA from Line 1 cells, 20 μg (lane 5). Probed with 32PO4 random hexamer primed, labeled cDNA insert 16Ab34.

Fig. 2.

Northern blot analyses of RNA samples from various murine cells and tissues. Poly(A)+-containing RNA from thymus, 5 μg (lane 1); uterus, 5 μg (lane 2); skin, 5 μg (lane 3) or Line 1 cells, 5 μg (lane 4); or total RNA from Line 1 cells, 20 μg (lane 5). Probed with 32PO4 random hexamer primed, labeled cDNA insert 16Ab34.

Another RNA blot was probed with a 227 bp sequence from murine β4 cDNA extending from base 3079 in the human sequence (Hogervorst et al., 1990). The same mRNA sizes were found and, in addition, the mRNA from intestines was shown to contain the 5.9 kb β4 mRNA (data not shown).

Cellular distribution of β4

In order to determine the cellular distribution of β4 expression in various organ sites, 125I-labeled mAb 346–11A was injected i.v. into animals and the microdistribution of antibody analyzed by tissue autoradiography. Since this is an unusual method of antigen detection, several control experiments were done. Data documenting the specificity of deposition of 125I-labeled mAb 346–11A in the mouse uterus are shown in Fig. 3. Animals injected with 5 μg of 125I-labeled mAb 11A show specific accumulation of mAb in the small vessels of uterus and along the basal cells of the luminal epithelium (Fig. 3A). If 1 mg of unlabeled mAb 11A is co-injected, an almost complete loss of signal is observed (Fig. 3B). In contrast, if 1 mg of unlabeled control mAb 14 is coinjected, no competition is noted (Fig. 3C) and if 125I-labeled control mAb 135–14 is injected, no localization in blood vessels is seen (Fig. 3D). These controls for the specificity of 125I-labeled mAb 11A deposition in other organs show identical competition patterns (data not shown), indicating specific detection of β4 by this method. Three other rat mAb have been studied in this manner. mAb 273-34A to thrombomodulin (Kennel et al., 1990), mAb 133–13A to CD44, and mAb 135–13C to the integrin subunit (in preparation), all give unique distribution patterns, indicative of the antigen distribution.

Fig. 3.

Autoradiographic analysis of tissue sections of uterus from mice treated with 5 μg of 125I-labeled mAb (12,300 counts/min per ng) alone (A) or mixed with 1 mg unlabeled mAb 346–11A (B) or with control mAb 135–14 (C). Mice treated with 5 μg of 125I-labeled control mAb 135–14 (13,200 counts/min per ng) (D).

Fig. 3.

Autoradiographic analysis of tissue sections of uterus from mice treated with 5 μg of 125I-labeled mAb (12,300 counts/min per ng) alone (A) or mixed with 1 mg unlabeled mAb 346–11A (B) or with control mAb 135–14 (C). Mice treated with 5 μg of 125I-labeled control mAb 135–14 (13,200 counts/min per ng) (D).

Autoradiography experiments using 125I-labeled mAb 346–11A to the β4 subunit are shown in Fig. 4. The spleen (Fig. 4A) has an arborizing pattern of small blood vessels (venules) apparent in the B cell follicles of the splenic white pulp. Only the central arteriole is stained in the T cell areas of the white pulp. No staining occurs in the red pulp. In the lung and adjacent tissue (Fig. 4B), abundant label is found in the walls of small pulmonary arteries and veins, in peribronchiolar arterioles and venules, and in the perineurial sheaths of large mediastinal nerves. The trachea is labeled in submucosal blood vessel walls and in basal portions of the mucosal epithelium (data not shown). In the thymus (Fig. 4C) label is present in the walls of small blood vessels (venules) and in portions of the capsule.

Fig. 4.

Autoradiographic analyses of tissue sections from mice injected with 10 μg of 125I-labeled mAb 346–11A (8,200 counts/min per ng) and killed 24 h after injection. Tissues were fixed in paraformaldehyde and embedded in paraffin prior to sectioning and overlaying with photographic emulsion. Approximate magnification, ×16. Spleen (A), lung (B), thymus (C), uterus (D), ovary (E), liver (F), intestine (G), intestine, Peyer’s patch (H), kidney (I), pancreas (J), esophagus (K) and skin (L).

Fig. 4.

Autoradiographic analyses of tissue sections from mice injected with 10 μg of 125I-labeled mAb 346–11A (8,200 counts/min per ng) and killed 24 h after injection. Tissues were fixed in paraformaldehyde and embedded in paraffin prior to sectioning and overlaying with photographic emulsion. Approximate magnification, ×16. Spleen (A), lung (B), thymus (C), uterus (D), ovary (E), liver (F), intestine (G), intestine, Peyer’s patch (H), kidney (I), pancreas (J), esophagus (K) and skin (L).

Label in the uterus (Fig. 4D) is found in the walls of blood vessels in the tunica muscularis and in the basal portion of the luminal and glandular epithelium. Label is also present in the basal portions of the cervical epithelium. Sections of the ovary (Fig. 4E) contain labeling in the walls of small blood vessels and on portions of the capsule. Large blood vessels in the surrounding fatty tissue are also labeled. In the liver (Fig. 4F), the walls of portal blood vessels and bile ducts are heavily labeled but no l25I-labeled mAb is found in the central veins.

In the gastrointestinal tract (Fig. 4G and H) the label occurs in the walls of small blood vessels in the lamina propria/submucosa and the tunica muscularis. Label is also seen in portions of the crypt epithelium in the duodenum and in epithelial cells at the tips of small intestinal villi. The entire kidney capsule (Fig. 4I) is heavily labeled. The staining of renal blood vessels is variable with location and size of the vessel. The outer surfaces of interlobar and arcuate arteries are labeled (tunica adventitia) while the label covers the entire wall of the corresponding veins. Patchy staining is also seen in the collecting ducts of the medullary rays and in the basal portion of the epithelium lining the renal pelvis.

Small blood vessels only are labeled in the pancreas (Fig. 4J).

Heavy label accumulation is noted in basal layers of the esophageal mucosa and in large vessels of the surrounding muscle (Fig. 4K). Labeled antibody in the skin (Fig. 4L) is found in small blood vessels throughout the dermis. There is patchy staining on the outer margins of some hair follicles; however, the basal epithelium does not show a major accumulation of mAb at this low antibody dose. Other tissues show significant accumulation of 125I-labeled mAb 346–11A in particular sites. In the lymph node, the radiolabel is localized to small blood vessels (venules) and to portions of the subcapsular sinus. In the heart (data not shown), there is a diffuse and abundant radioactive label on the valvular endothelium and a mild, diffuse label on the endocardium of the atria and ventricles. Patchy staining is present on the epicardial (outer) surface of the ventricles. The intimal surfaces of the pulmonary veins and arteries are labeled but no stain is found on the aorta.

In general mAb 346–11A is on the endothelial surface of small to medium-sized arteries and veins throughout the body. It does not bind to capillary vessels, to the central veins in the liver, or to the intimal surface of the aorta. Detection of β4 expression in some epithelia by this method requires higher doses of 125I-labeled mAb to saturate the intervascular sites and allow mAb to extravásate to the epithelial sites. Experiments with 100 μg of 125I-labeled mAb detect high expression of 4 in the basal cells of skin (data not shown). In addition, mAb 346–11A also binds to basal portions of epithelia in eye, trachea, esophagus, gastrointestinal tract and uterus, as well as intrahepatic bile ducts and renal collecting ducts. At this resolution, it is not possible to determine if the mAb binds to the basal surface of the endothelium adjacent to the basement membrane as has been shown for its expression at epithelial sites (Sonnenberg et al. 1990c).

Previous studies on the distribution of the β4 subunit utilizing standard immunohistochemistry indicate that it is epithelial cell-specific (Sonnenberg et al., 1990a; Kajiji et al., 1989; Tamura et al., 1990). Using a different method to detect β4 expression, we show that this subunit is also expressed on endothelial cells of a unique subset of blood vessels. Quantitative assay for β4 distribution is in general agreement with published results on β 4 distribution (Sonnenberg et al., 1990a; Kennel et al., 1986) and with data for ‘“I-labeled mAb deposition in vivo when data at different doses are evaluated. This is consistent with the contention that although α 6 can associate with either & or β 4 the only a subunit that is co-expressed with β 4 is (Sonnenberg et al., 1990a). Combined immunohistochemistry and in situ hybridization may allow direct identification of β 4 expression in particular cell types of tissues.

The determination of β 4 distribution by deposition of 125I-labeled mAb in vivo is a new approach to antigen identification. We have shown that this deposition is specific by doing competition experiments (Fig. 3), and by control 125I-labeling mAb distributions. Several mAbs have been evaluated in this manner, each giving unique microdistribution (Kennel et al., 1990; Kennel et al., 1991), indicating the antigen specificity of the reaction. This method is non-quantitative and, at low mAb doses, emphasizes expression in the vascular space where antigen is accessible to the antibody (Kennel et al., 1991; Kennel et al., 1990). Previous immunohistochemistry studies (Sonnenberg et al., 1990a) have shown that β 4 is prevalent in basal epithelium of colon, skin, trachea, esophagus and linings of peripheral nerves. These locations are also identified by deposition of 125I-labeled mAb, although deposition in epithelia is light at low mAb dose. Epithelial expression is more easily detected at higher mAb dose (data not shown). In addition, l25I-labeled mAb is heavily deposited along the endothelium of intermediate-sized blood vessels and in the epithelia of uterus and ovaries (tissues not previously studied). At the dose of mAb used, most of the deposition is noted in particular subsets of endothelia. The selectivity of mAb deposition emphasizes the specificity. For example, lung, which contains one of the largest concentrations of capillaries, shows mAb in medium-sized vessels only. It has not been determined whether deposition differs between bronchial and pulmonary veins. In the liver, portal blood vessels are stained but there is no label in the central veins or sinusoids. In the spleen, only the small vessels of the white pulp are stained, and there is no mAb deposition in the red pulp, which has sinusoidal endothelium. These sites of expression are difficult to detect with standard immunohistochemistry, probably due to the low levels of β4 present.

Several artifactual complications can be associated with this type of experiment. First, it is possible that the 125I-labeled mAb is dehalogenated and free I- is being detected. The specificity of deposition argues against this as a major complication. It is possible that certain sites of β4 expression in the body (i.e. liver) are missed due to rapid dehalogenation at the site of deposition. However, this hypothesis cannot explain the lack of label in lung capillaries on the red pulp of spleen, since other l25I-labeled mAbs have been effectively localized at these sites (Kennel et al., 1991; Kennel et al., 1990). Furthermore, another mAb has been shown to localize in the sinusoids at the liver (data not shown), indicating that dehalogenization is not a major problem under these conditions. Another possibility is that mAb is aggregated in immune complexes. Again, the observed distribution of 125I is inconsistent with this hypothesis, since aggregates accumulate in kidney, liver and the red pulp of the spleen, and this distribution is not seen with 125I-labeled mAb 346-11A. A final concern is that the mAb is binding to a circulating cell (lymphocyte or platelet) and that these cells show specific localization. The β4 subunit has not been detected on circulating blood cells. In addition, 125I-labeled mAb to (prevalent on platelets) gives a completely different distribution (data not shown), consistent with that expected for immune complex deposition.

The data indicate that the endothelium of intermediate-sized vessels is a site of β4 expression in the mouse. Quantitative assay of β4 concentration in mouse organs (Kennel et al., 1986) correlates in general with deposition of 125I-labeled mAb 346–11A; however, these quantitative data do not determine if the majority of β4 in these organs is on epithelium, or endothelium. Recently, β 4 has been shown to be associated with hemidesmosomes in epidermis and peripheral nerves (Stepp et al., 1990; Carter et al., 1990; Sonnenberg et al., 1990b), indicating a role in adhesion to basement membrane and intermediate filaments. Since hemidesmosomes are not detected in blood vessels, some other association may be operative. It has been shown conclusively that α6β1 is a laminin receptor (Sonnenberg et al., 1988; Aumailley et al., 1990) but evidence for that function for α6β1 is mixed (Sonnenberg et al., 1990a; Lotz et al., 1990). It may be that α6β3 has slightly different functions when expressed on epithelium versus endothelium or it is still possible that A6β4 binds to a specialized variant of laminin. If this is true, one would predict high concentrations of the specialized laminin in the basement membrane of the intermediatesized vessels and also in basement membrane of skin, colon and uterus. It is still necessary to keep open the idea that α6β4 may function in cell-cell interaction as well as adherence to basement membrane. Recent evidence (Tamura et al., 1990; Hogervorst et al., 1990) indicates a number of alternatively spliced mRNAs for β4 as well as for (Hogervorst et al., 1991). It is possible that these different messages code for proteins with variant functions, particularly in the cytoplasmic domain. Northern blot analyses of various tissues detected mRNA of only one size for β4, with three different probes (data not shown). It is possible that minor amounts of spliced mRNA would not be detected or that small differences, i.e. 50 bases, would go undetected. Whether or not different forms and functions exist for β4, it is clear that endothelial cells of certain specialized blood vessels express a significant amount of the β4 subunit.

We thank Drs. M. Terzaghi-Howe and R. J. M. Fry for review of this manuscript. C. Rains and N. Crowe were instrumental in manuscript preparation. David Blazes helped in the subcloning and sequence analyses of 16Ab34, and J. Wesley provided histochemistry support.

The submitted manuscript has been authored by a contractor of the U.S. Government under contract No. DE-AC05-840R21400. Accordingly, the U.S. Government retains a nonexclusive, royalty-free license to publish or reproduce the published form of this contribution, or allow others to do so, for U.S. Government purposes.

Research was sponsored by the Office of Health and Environmental Research, United States Department of Energy, under Contract DE-AC05-840R21400 with the Martin Marietta Energy Systems, Inc.

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