Localization of the Transmembrane 4 Superfamily (TM4SF) Member PETA-3 (CD151) in Normal Human Tissues: Comparison with CD9,CD63,and α5β1 Integrin

Abstract

It has recently been shown that several members of the tetraspan superfamily, including CD9 and CD63, associate with each other and with β1 integrins. In this study, we examined the distribution of a recently identified tetraspan, PETA-3 (CD151), and of CD9, CD63, α5β1, and the integrin β1 chain in normal human tissues by the indirect immunoperoxidase and alkaline phosphatase-anti-alkaline phosphatase techniques. PETA-3 showed a broad distribution and was expressed by endothelium, epithelium, Schwann cells, and dendritic cells and by skeletal, smooth, and cardiac muscle. Expression in skin was mostly restricted to the basal cells of the epidermis and was downregulated on differentiation. In the small intestine, PETA-3 was expressed by crypt and villous enterocytes with a mostly basolateral distribution, but was not detectable on the brush border. CD9 was expressed on the plasma membrane of enterocytes in crypts and at the bases of the villi whereas CD63 demonstrated a unique granular appearance concentrated in the apical cytoplasm below the brush border. The findings of this study show co-localization of PETA-3 with CD9, CD63, α5β1, and β1 in particular tissues, demonstrating that tetraspan/integrin complexes may occur. However, the lack of co-localization of these antigens in other tissues also implies distinct roles for these molecules.

Keywords

PETA-3 CD151 CD9 CD63 TM4SF integrin

Recently, a cDNA clone encoding a 27-kD protein PETA-3 (platelet endothelial tetraspan antigen-3) was isolated from a library constructed from the mega-karyoblastic cell line MO7e (Fitter et al. 1995). The clone encoded an open reading frame of 253 amino acids and sequence comparisons revealed 25–30% amino acid identity with several members of the recently described transmembrane 4 superfamily (TM4SF), also known as tetraspans. Subsequently, PETA-3 was independently cloned from the adult T-cell leukemia cell line SF-HT by Hasegawa et al. (1996) and was named SFA-1 (SF-HT-activated gene 1). PETA-3, or SFA-1, expression was shown to be upregulated after transformation of SF-HT cells by human T-cell leukemia virus Type 1. PETA–3 was assigned CD151 at the VIth International Leucocyte Differentiation Antigen workshop (Ashman et al. in press).

Members of the TM4SF are characterized by four highly conserved hydrophobic regions that are believed to be membrane-spanning which show little homology with other proteins containing four transmembrane domains (Wright and Tomlinson 1994; Horejsi and Vlcek 1991). Also characteristic of tetraspan proteins is the presence of a large divergent extracellular loop between the third and fourth membrane-spanning domains, which has been suggested to define the functional specificity of the particular tetraspan. Members of the TM4SF have been identified in a diverse range of organisms, including Schistosoma (Wright et al. 1990), Drosophila (Kopczynski et al. 1996), and humans (Wright and Tomlinson 1994). This evolutionary conservation of tetraspans suggests an integral role for these molecules in cell biology.

Although the biochemical functions of the TM4SF are unknown, several studies using monoclonal antibodies (MAbs) have shown tetraspans to mediate a broad range of biological responses. Anti-CD9 and anti-PETA-3 MAbs are able to induce platelet activation and mediator release by crosslinking of FcgammaRII, the low-affinity IgG receptor (Roberts et al. 1995; Ashman et al. 1991; Worthington et al. 1990). More recent studies in platelets have demonstrated intrinsic signaling capacity for CD9, with anti-CD9 F(ab')₂ fragments inducing phosphorylation of the tyrosine kinase syk (Ozaki et al. 1995). Signal transduction events have also been demonstrated in B-cells, granulocytes, and monocytes by anti-CD53 MAbs (Olweus et al. 1993). MAbs against CD9 and CD81 can cause homotypic adhesion of pre-B-cells (Masellis-Smith et al. 1990; Takahashi et al. 1990). Increased adhesion of neutrophils to endothelium (Forsyth 1991) and adhesion of pre-B-cells to bone marrow fibroblasts through the modulation of fibronectin receptors has also been demonstrated for anti-CD9 MAbs (Masellis-Smith and Shaw 1994). A potential role for tetraspans in modulating adhesion events has been further supported by the finding that several members are associated with integrin complexes. Initially, CD9 was demonstrated to associate with the αIIb/β3 complex in platelets (Slupsky et al. 1989), and subsequent studies have highlighted associations with integrins of the β1 subfamily. For example, immunoprecipitation studies have shown CD9 to be associated with both α4β1 and α5β1 in the pre-B-cell line NALM-6, whereas in the erythroleukemia-derived cell line HEL, CD9 was associated only with α4β1 (Rubinstein et al. 1994). CD63 has been demonstrated to associate with α3β1 and α6β1 integrins in HT1080 fibrosarcoma cells and in transfected K562 cells expressing either the α3 or α6 chain (Berditchevski et al. 1995). Furthermore, CD9, CD63, and CD81 were demonstrated to associate with each other and α3β1 to form tetraspan/integrin and tetraspan/tetraspan/integrin complexes (Berditchevski et al. 1996). Similarly, PETA-3 has recently been shown to associate with α5β1, and CD63 in MO7e and HEL cells (S. Fitter, unpublished data) and CD9 and the integrin β1 chain in platelets (P. Sincock, unpublished data). The physical association of several tetraspan members with integrins suggests a possible role for tetraspans in modulation of integrin function.

Previous studies using MAb 14A2.H1 have shown expression of PETA-3 on platelets, endothelium, some myeloid leukemia cells, and epithelium of tonsil sections (Ashman et al. 1991). Furthermore, studies of PETA-3 mRNA distribution by Northern blotting analysis showed mRNA expression in all tissue homogenates examined, with the exception of brain (Fitter et al. 1995). Because PETA-3 is expressed by endothelium, it was unclear whether or not the vasculature component of the tissues examined accounted for their positivity by Northern analysis. Furthermore, the association of PETA-3 with α5β1, CD9, and CD63 by biochemical studies suggests that these antigens may be co-expressed with PETA-3 in particular tissues. This study investigated the tissue localization of the newly described PETA-3 and the possibility that it is co-localized with CD9, CD63, α5β1, and β1 chain in normal human tissues.

Materials and Methods

Tissues

Normal adult human tissue specimens were obtained with approval from the Royal Adelaide Hospital Ethics Committee. Tonsil and gut were obtained through surgical procedures and embedded within 1 h. Postmortem tissues, including brain, skeletal muscle, cardiac muscle, skin, kidney, liver, lung, pancreas, spleen, thyroid, and adrenal gland, were obtained within 6 hr of death. Solid tissues were placed in Tissue-Tek OCT compound (Miles; Elkhart, IN) and frozen by immersion into isopentane precooled in liquid nitrogen. Skeletal and cardiac muscle were not embedded in OCT but were plunged directly into liquid nitrogen and stored at — 70C. Fresh frozen air-dried sections (5 μm) were fixed in 95% ethanol for 10 min at 4C and washed with three changes of cold (4C) PBS before staining by the indirect immunoperoxidase technique. Peripheral blood was collected in heparin and bone marrow mononuclear cells (BMMNCs) were obtained and Ficoll-purified as previously described (Simmons et al. 1994). Blood and cytocentrifuge slides were allowed to air-dry and were stored at — 70C before staining with the alkaline phosphatase–anti-alkaline phosphatase (APAAP) technique.

Monoclonal Antibodies

The anti-PETA-3 MAb 11B1.G4 (IgG_2a) was raised by fusion of splenocytes from Balb/c mice immunized with MO7e megakaryoblastic leukemia cells with the X-63.Ag8.653 myeloma cell line. The resultant hybridoma supernatants were screened on FDCP1 cells transfected with PETA-3 cDNA (Fitter et al. 1995). Anti-CD9 MAb 1AA2.H9 (IgG¹) (Cole et al. 1989) and anti-CD63 MAb 12F12.1G2 (IgG¹) (Zannettino et al. 1996) were raised as described. The MAb 61.2C4 (IgG¹) against the β1 integrin chain (unpublished) was a kind gift from Dr. J. Gamble (Hanson Centre for Cancer Research) and the anti-α5β1 MAb PHM2 (IgG¹) (Hancock et al. 1983; Becker et al. 1981) was kindly supplied by Dr. Randall Faul (Renal Unit, Royal Adelaide Hospital). Isotype-matched negative control MAbs 1B5 (IgG¹), anti-Giardia (G. Mayrhofer, unpublished) and Sal-1 (IgG_2a), anti-Salmonella (O'Connor and Ashman 1982) were also included in all experiments.

Immunohistochemistry

All solid tissues were examined by the indirect immunoperoxidase technique, and peripheral blood smears and bone marrow aspirates were examined by the APAAP technique. To block Fc-mediated binding, MAb culture supernatant was supplemented with 10% heat-inactivated normal rabbit serum before application to sections. For indirect immunoperoxidase staining, sections were allowed to incubate for 1 hr at 4C with MAb. After washing in three changes of cold PBS, sheep anti-mouse immunoglobulin (Ig)-peroxidase (Amersham; Poole, UK) diluted 1:20 in 10% NRS was applied and incubated for 1 hr at 4C. After further washes, sections were brought to room temperature and peroxidase activity was detected by incubation with 1.3 mM 3,3′-diaminobenzidine tetrahydrochloride (Sigma; St Louis, MO) containing 0.07% hydrogen peroxide (Sigma). Sections were then washed in PBS, counterstained with Mayer's hematoxylin, dehydrated through alcohol and safsolvent (Ajax Chemicals, Australia) gradients, and mounted with Depex (BDH Gurr; Sydney, Australia). APAAP staining was performed as previously described (Ashman et al. 1991), with the exception that cells were fixed with methanol.

Table 1

Immunohistochemical localization of PETA-3, CD9, CD63, α5β1, and the β1 integrin chain in normal human tissues a

Tissues	PETA-3	CD9	CD63	α5β1	β1 Chain
Skin
Stratum basale	++++	++++	-	++	+++++
Stratum spinosum	+/-	+++++	+/-	+/-	+
Stratum	-	+++++	+/-	+/-	-
granulosum
Gut
Epithelium	++	+++	++	-	+++
Lamina propria	++	+++++	+	+++	+++++
IELs	-	+	++	-	+
Connective tissue
Fibroblasts	-	++++	+++	-	++
Lung	+++++	++++	++	+++	+++++
Kidney
Glomeruli	+++++	++	++	+	+++++
Proximal/distal	++	+	++	-	+++
tubules
Collecting ducts	+	+++++	++	-	++++
Liver
Hepatocytes	+	+	-	-	-
Bile ducts	++++	++++	+++	++	++++
Sinusoids	+++++	+	++++	-	+++
Pancreas
Acini	+	-	+++++	-	-
Ducts	++	-	++	++	++
Cerebral cortex
Vasculature	++++	++++	+++	+++	+++++
Gray matter	-	+	-	-	-
White matter	-	+++++	-	-	-
Spleen
White pulp	+	+	+	-	+
Red pulp	+++	+++	+++	+	+++++
Central arteries	+++++	+++++	+/-	+++	+++++
Tonsil
Squamous	+++	+++++	++	+++	+++++
epithelium
Germinal center	++++	+++	++	+	+++
HEVs	++++	++++	+++	++	+++++
Circulatory
Arteries	+++++	++++	++	++	+++++
Veins	+++++	++++	++	++	+++++
Capillaries	++++	+++	++	++	++++
Perivascular	+++++	+++++	+/-	+	+++++
smooth muscle
Peripheral blood
Red blood cells	-	-	-	-	-
Lymphocytes	-	+/-	-	+/-	+
Neutrophils	-	-	+++++	-	+++
Monocytes	-	+	++	+	+
Platelets	++++	+++++	+++	++	+++++
Bone marrow MNCs
Megakaryocytes	+++++	+++++	++++	+++	++++
Skeletal muscle	+++	++	+	-	++++
Peripheral nerve
Perineurium	+++++	+++++	+	+	+++++
Endoneurium	-	-	-	-	-
Schwann cells	++++	++++	+	++	+
Cardiac muscle	+++++	-	++	+	+++++
Adrenal
Cortex	++	++	+++++	-	++++
Thyroid
Follicular cells	+	++	++++	-	-
Parafollicular cells	++	++	+++	-	+++

Level of staining was scored from + (weakly positive) to +++++ (strongly positive). MNCs, mononuclear cells; IELs, intraepithelial lymphocytes; HEVs, high endothelial venules.

Results

All tissues were examined for expression of PETA-3 using MAb 11B1.G4 in the indirect immunoperoxidase assay. Adjacent sections were stained with MAbs against CD9 (1AA2.H9), CD63 (12F12.1G2), the β1 integrin family (anti-β1 MAb 61.2C4), and the α5β1 integrin (PHM2). The findings are summarized in Table 1. Isotype-matched negative control MAbs were included in all experiments as specificity controls. Apart from the endogenous peroxidases of eosinophils, alveolar macrophages, and Kupffer cells, no other cells in any of the tissues stained with these MAbs by the indirect immunoperoxidase technique. One exception was tonsillar follicular dendritic cells, which showed low levels of binding of the IgG_2a control Sal-1. No staining of peripheral blood/bone marrow cells was seen with negative control MAbs by the APAAP technique.

Tissue Distribution of PETA-3, CD9, CD63, α5β1, and the Integrin β1 Subfamily

Vascular endothelium was stained strongly with MAbs against PETA-3, CD9, and β1. Perivascular smooth muscle and several connective tissue components were also positive. A similar distribution was seen for α5β1. However, the degree of staining was much weaker and connective tissue components failed to react. CD63 was identified on endothelium and connective tissue components, but was not detected on smooth muscle. The following descriptions are of PETA-3, CD9, CD63, α5β1, and β1 integrin localization within the tissues examined. The results obtained are summarized in Table 1.

Figure 1

Immunohistochemical localization of tetraspans and β1 integrins in human skin and small intestine. (A) Staining of basal kerati-nocytes by anti-PETA-3 MAb. Note the more intense staining adjacent to the basal lamina. (B) Staining of basal keratinocytes was also seen for β1 integrins and, to a lesser degree, for (C) α5β1. (D) CD9 was strongly expressed by keratinocytes throughout the epidermis. (E) PETA-3 stained the basolateral membranes of enterocytes on the villi. Within the lamina propria, smooth muscle and capillaries were also stained. (F) β1 integrins showed a localization similar to that of PETA-3. (G) CD9 is expressed on the basolateral membranes of enterocytes, and expression is lost by epithelial cells as they ascend the villus. (H) CD63 was localized in a granular distribution below the brush border. Some intraepithelial lymphocytes were also stained. (I) α5β1 was expressed by smooth muscle fibers in the lamina propria. Bars = 40 μm.

Figure 2

Immunohistochemical localization of PETA-3 in blood vessels, including specialized endothelium, in tonsil, and in BMMNCs. (A) Muscular artery within kidney showing expression of PETA-3 by the endothelium and vascular smooth muscle. (B) Kidney glomerulus, (C) hepatic sinusoids, (D) cerebral capillaries, (E) central arterioles of the splenic white pulp, and (F) tonsil HEVs showed similar intensities of staining for PETA-3. (G) Low-power view showing immunohistochemical localization of PETA-3 in a section of human tonsil. (H) Staining of follicular dendritic cells in germinal centers of human tonsil. (I) APAAP staining of BMMNCs with MAb 11B1.G4, demonstrating intense staining of a megakaryocyte. Platelets were also positive for PETA-3. Bars: A = 150 μm; B–H = 75 μm; E,F,I = 40 μm; G = 375 μm.

Skin . PETA-3 was expressed on the cell membrane of the basal keratinocytes. Expression was strongest on the membrane adjacent to the basal lamina (Figure 1A). The β1 integrin chain was also highly expressed on the membrane of basal keratinocytes (Figure 1B). In contrast, α5β1 integrin was expressed at low levels by keratinocytes throughout the epidermis (Figure 1C). Anti-CD9 MAb produced intense staining of all keratinocytes (Figure 1D) and, in further contrast to PETA-3, did not stain the cell membrane of basal keratinocytes that contacts the basal membrane. Anti-CD63 stained keratinocytes very weakly, although melanocytes stained strongly, as has been previously described (Hotta et al. 1988).

Small Intestine. PETA-3 was expressed on the basolateral membranes of the columnar epithelial cells of the villi (Figure 1E) and crypts. Smooth muscle cells within villi and the muscularis mucosa were also positive for PETA-3. β1 integrins were present on the basolateral membranes of epithelium in crypts and on the villi. However, the anti-β1 MAb also diffusely stained the basal cytoplasm (Figure 1F). Smooth muscle cells within villi and the muscularis mucosa were also strongly positive for β1 integrin. CD9 (Figure 1G) was expressed on the basolateral membrane of immature enterocytes in the crypts and on the bases of the villi. The anti-CD9 MAb also stained smooth muscle. CD63 was also detected on crypt and villous epithelium but, in contrast to PETA-3 and CD9, CD63 had a granular appearance and was localized to the apical cytoplasm below the brush border (Figure 1H). α5β1 integrin was detected only on smooth muscle fibers within the villi (Figure 1I) and the muscularis mucosa. Staining of some intraepithelial lymphocytes (IELs) with MAbs against CD9, CD63, and β1 integrin was also observed.

Connective Tissue. Within the dense connective tissue of the deep dermis, fibroblasts were stained by MAbs to CD9, CD63, and the β1 integrin chain. PETA-3 and α5β1 were not detected on fibroblasts. In the superficial dermis, cells with irregular processes were stained strongly by anti-CD63 MAb and at lower levels by MAb against PETA-3, CD9, and β1. No detectable staining of these cells was seen with anti-α5β1. Although these cells may be macrophages and/or mast cells, other histochemical techniques would be required to confirm their identity.

Lung. Within the lung parenchyma, smooth muscle associated with bronchi, bronchioles, and blood vessels, epithelium, and alveolar pneumocytes were stained strongly by MAb against PETA-3, CD9, and the β1 integrin chain α5β1 was also present on these cell types, although staining was less intense. CD63 was present on pneumocytes and airway epithelium, but staining was less intense than with the other MAbs. Endothelial cells of alveolar capillaries and larger blood vessels were stained by all MAbs tested. Alveolar macrophages showed some staining with negative control MAbs. Therefore, the specificity of staining of these cells could not be determined.

Kidney. Strong staining with MAb against PETA-3 and β1 integrin was seen on periarteriolar smooth muscle and endothelial cells of blood vessels and glomeruli (Figures 2A and 2B). Staining was weaker for CD9, CD63 and α5β1 on glomerular endothelium. Epithelium of the proximal and distal convoluted tubules was stained weakly with MAbs against β1 integrin, PETA-3, and CD63. Very weak staining of these tubules was observed with anti-CD9, and no detectable staining was seen with anti-α5β1. In contrast to the staining observed with other tetraspan MAbs, anti-CD9 strongly stained epithelial cells of collecting ducts. The β1 integrin chain was also strongly stained on collecting ducts, but no staining of α5β1 was detected.

Liver. Endothelium in hepatic sinusoids stained strongly positive for PETA-3 (Figure 2C), CD63, and β1 integrin chain, whereas staining for CD9 and α5β1 was much weaker. Columnar epithelium of intrahepatic bile ducts was stained with all MAbs examined. The cell membranes of hepatocytes also showed weak staining by MAbs against PETA-3, CD9, and the β1 integrin chain. Kupffer cells showed weak staining with negative control MAbs. Therefore, the specificity of staining of these cells could not be determined.

Pancreas . Acinar cells and pancreatic islets were either unstained or lightly stained for most MAbs tested. However anti-CD63 staining of these cells was very strong. The columnar epithelium of intralobular excretory ducts showed diffuse staining by MAbs against PETA-3, CD63, the β1 integrin chain, and α5β1, but CD9 was not detected.

Cerebral Cortex . Vascular endothelium was positive for PETA-3 (Figure 2D), CD63, α5β1, and β1. Nerve bodies in the gray matter and myelinated fibers in the white matter were not stained. Anti-CD9 MAb also stained endothelium and, in addition, the white matter showed strong diffuse staining of the neuropil, as previously described (Rossler et al. 1992).

Spleen . Within adult spleen, lymphocytes in the white pulp were unstained by all MAbs. Low-level staining was seen with anti-PETA-3, CD9, CD63, and β1 MAbs in the region of periarteriolar lymphoid sheaths and white pulp, which may have been staining of interdigitating cells. Central arterioles were stained strongly by anti-β1 MAb and less strongly by MAbs against PETA-3 (Figure 2E), CD9, and α5β1. Staining of central arterioles by anti-CD63 was barely detectable. Red pulp sinusoids and splenic cords were also stained by MAbs against PETA-3, CD9, CD63, α5β1, and the β1 chain.

Tonsil . Squamous epithelium, germinal centers, and high endothelial venules (HEVs) were stained by all MAbs tested. As in skin, expression of PETA-3 and β1 by epithelial cells was restricted to the basal layer, whereas strong staining for CD9 was observed on cell membranes throughout the epithelium. Staining of CD63 and α5β1 was weak and showed a similar distribution to that of CD9. Lymphocytes within both T-and B-cell zones were not stained by any of the MAbs tested. HEVs were stained moderately by anti-CD63 and were strongly positive for PETA-3 (Figure 2F), CD9, α5β1, and the integrin β1 chain. Follicular dendritic cells within germinal centers were stained by all MAbs tested. Staining was weak for α5β1 and CD63 and was stronger for PETA-3 (Figures 2G and 2H), CD9, and the β1 integrin chain. Although some staining was seen with the IgG_2a negative control Sal-1, the staining by anti-PETA-3 MAb 11B1.G4 (IgG_2a) was much stronger, and the IgG₁ anti-PETA-3 MAb 14A2.H1 also stained tonsillar follicular dendritic cells (data not shown), thus verifying the specificity of staining for these cells.

Peripheral Blood . PETA-3 staining was restricted to platelets only. MAbs against CD9, CD63, β1, and α5β1 also stained platelets. Several other hemopoietic cell types, including neutrophils, macrophages, and monocytes (see Table 1), were also stained by these MAbs as has been previously described (reviewed in Barclay et al. 1993).

Bone Marrow Mononuclear Cells . PETA-3 expression was mostly restricted to cells of the megakaryocytic lineage, which stained very strongly (Figure 2I). Immature cells of the neutrophil lineage showed weak staining. Infrequent small cells were also weakly stained, and further studies would be required to determine their nature. All of the other MAbs tested stained cells of the megakaryocytic lineage as well as many other cell types, as reviewed in Barclay et al. (1993).

Peripheral Nerve and Skeletal Muscle . Peripheral nerve fibers were identified within transverse sections of skeletal muscle. The perineurium of peripheral nerve fascicles was strongly positive for PETA-3 (Figure 3A), CD9, and β1 integrins, and was weakly positive for CD63 and α5β1. Staining with isotype-matched negative control MAb is shown in Figure 3B. The myelin sheaths and cell bodies around the nuclei of Schwann cells were strongly positive for MAbs against PETA-3 and CD9 and also showed lower levels of staining for CD63 and the β1 integrin chain. Transverse sections of skeletal muscle showed that PETA-3 (Figure 3A), CD9, CD63, and β1 integrin were expressed on the sarcolemma of striated muscle cells. α5β1 was not detected on skeletal muscle cells.

Figure 3

(A) Immunohistochemical localization of PETA-3 in a transverse section of skeletal muscle. PETA-3 was expressed on capillary endothelium and the sarcolemma of muscle cells. The perineurium and myelin sheaths of nerve fibers in peripheral nerv bundles were stained strongly with MAb against PETA-3. (B) Isotype-matched negative MAb staining of skeletal muscle. Bars = 90 mm.

Heart . Cardiac muscle showed staining for PETA-3 (Figure 4A) and β1 integrin (Figure 4B) on the sarcolemma of muscle cells and capillary endothelium. Some diffuse cytoplasmic staining of cardiac muscle cells was also seen with these MAbs. Anti-CD63 and α5β1 staining was much lower on cardiac muscle cells, and CD9 (Figure 4C) was restricted to the endothelium of blood vessels. Absence of staining in cardiac tissue with isotype matched negative control MAb is shown in Figure 4D.

Adrenal Cortex . Parenchymal cells of the zona glomerulosa and spongiocytes within the zona fasciculata were stained by anti-CD63 and β1 MAbs. PETA-3 and CD9 were also expressed by these cells. In each case, the MAbs stained the cell membranes and the periphery of cytoplasmic lipid droplets. Cells within the zona reticularis were also stained by these MAbs, although the pattern of staining on these cells was diffuse and cytoplasmic. Anti-CD63 stained spongiocytes strongly and cells of the zona reticularis very strongly. α5β1 expression was not detected on parenchymal cells in any of the zones of the adrenal cortex.

Figure 4

Immunohistochemical localization of (A) PETA-3, (B) β1 integrins, and (C) CD9 in cryostat sections of human cardiac muscle. Both PETA-3 and β1 integrins were strongly expressed by capillary endothelium and cardiac muscle cells. CD9 was restricted to the endothelium of capillaries only. (D) Isotype-matched negative MAb staining of cardiac muscle. Bars = 50 μm.

Thyroid . The follicular epithelium and parafollicular cells were stained by MAbs against PETA-3, CD9, and CD63. Anti-PETA-3 and CD9 MAbs stained the cell membranes of follicular epithelial cells, whereas staining with CD63 showed a granular appearance. Follicular epithelium was not stained by anti-β1 MAb, although β1 integrin was detected on the membrane of parafolicular cells with a distribution similar to that of PETA-3 and CD9. α5β1 was not detected on follicular epithelium or parafolicular cells.

Discussion

The association of tetraspan molecules with each other and with β1 integrins has suggested a role for these proteins in the modulation of integrin function (Berditchevski et al. 1995,1996; Rubinstein et al. 1994). PETA-3, like other members of the TM4SF, appears to share these associations. Complexes containing PETA-3 in association with CD63 and α5β1 in both MO7e and HEL cells (S. Fitter, unpublished data) have been shown. In addition, PETA-3 has been demonstrated to co-precipitate with CD9 and the integrin β1 chain from platelet lysates (P. Sincock, unpublished data). Although much emphasis has been placed on the characterization of these associations using cell lines, the distribution of PETA-3 in vivo and its co-localization with associated antigens in various tissues have not been studied in detail. Recently, it was shown that CD9, CD63, CD81, and α3β1 co-localized in some cells in tissue sections of skin, breast, tonsil, and colon (Berditchevski et al. 1996). Furthermore, co-immunoprecipitation experiments provided direct evidence demonstrating the existence of tetraspan/tetraspan/integrin complexes involving these molecules (Berditchevski et al. 1996). Because PETA-3 has also been demonstrated to be involved in such complexes, the aim of our investigation was to determine the tissue distribution of PETA-3 and to compare systematically the localization of known associated molecules, including CD9, CD63, and the α5β1 integrin. PETA-3 was readily detected on vascular endothelium and smooth muscle in all tissues examined. Fibroblasts and some other undefined cells within connective tissues also expressed PETA-3, CD9, CD63, and β1 integrins, but α5β1 was undetectable. The widespread expression of these antigens throughout connective tissue probably reflects their interaction with components of the extracellular matrix such as collagen, laminin, and fibronectin.

Expression of PETA-3 by vascular endothelium was constant throughout all tissues examined, including regions of specialized endothelium (e.g., glomeruli, HEVs, hepatic sinusoids, alveolar capillaries, and capillaries constituting parts of the blood-brain barrier). The widespread expression of PETA-3 in endothelium suggests that its primary role may be in the maintenance of vascular integrity. This could be achieved through cooperation with integrins in modulating adhesion events with extracellular matrix, between endothelial cells, or with formed elements of blood. In addition, the widespread expression of PETA-3 suggests that endothelial activation by locally produced proinflammatory cytokines does not regulate the level of expression. Such regulation of expression was seen with CD63, a known marker of endothelial activation (Vischer and Wagner 1993), which was not detected within central arteries of spleen yet was expressed within HEVs of the tonsil and most other vasculature. Interestingly, PETA-3 was expressed on the vasculature of brain tissue, although PETA-3 mRNA had not been previously detected by Northern analysis (Fitter et al. 1995). CD63, α5β1, and β1 were also restricted to the vasculature in brain tissue, whereas CD9 was expressed by other tissue components as well. Expression of CD9 in the brain parenchyma has been previously described (Rossler et al. 1992), and results of the present study are similar. Interestingly, the restriction of the β1 integrin chain to the vasculature in brain suggests that CD9, unlike the other tetraspans, may be predominantly associated with another integrin β-chain or that it has an integrin-independent function in these cells.

Throughout most tissues in this study, the tetraspans investigated showed similar patterns of distribution. The co-localization of the tetraspan antigens with each other and with β1 integrins, in some cases α5β1, is consistent with the concept that they can associate to form tetraspan/integrin or tetraspan/tetraspan/integrin complexes. PETA-3 did co-localize with α5β1 in several tissues examined, especially those that showed very high levels of PETA-3 expression. However, the stronger staining of β1 integrin throughout most tissues indicated the presence of other β1 integrins. Therefore, PETA-3 may associate with other β1 integrins in such cells.

PETA-3 was expressed at relatively high levels by cardiac muscle cells and was also expressed on striated skeletal muscle. The lack of co-localization of PETA-3 with α5β1 in muscle cells again suggests that other β1 integrins may associate with PETA-3 in vivo. Because it has been demonstrated that CD9 associates with more than one β1 integrin, i.e., α4β1 and α3β1, it is possible that PETA-3 also associates with several β1 integrins.

PETA-3 expression was also observed in a number of epithelia. In the skin and tonsil, staining was restricted to the basal layers of the stratified epithelia and exhibited polarity, with brightest staining adjacent to the basal membrane. In contrast, CD9 was expressed by all keratinocytes in skin and showed an opposite polarity in cells of the stratum basale. The presence of PETA-3 close to the basement membrane suggests that it is involved in anchoring of cells to the basal lamina. The loss of PETA-3 by keratinocytes as they leave the proliferative compartment and lose contact with the basal lamina further supports this hypothesis. In contrast, CD9 may be primarily involved in interactions between keratinocytes, as suggested by its expression by keratinocytes throughout the epidermis and decreased expression on the basal membrane in cells of the stratum basale. β1 integrin expression was also restricted to the basal layers, suggesting that association with PETA-3 is involved in anchoring to the basement membrane.

Expression of PETA-3 by simple columnar or cuboid epithelia also demonstrated a basolateral distribution, especially in villous and crypt enterocytes of the small intestine. Whereas α5β1 expression was not detected on these cells, the β1 integrin chain was present in a similar polarized distribution, again providing further evidence that PETA-3 may associate with other members of the β1 subfamily. The possibility of PETA-3 associations with other β1 integrins is presently being investigated. The basolateral distribution of PETA-3, CD9, and the β1 chain in epithelial cells implies that they may be involved in interactions of the cells with the basement membrane and possibly in intercellular adhesion. The expression of CD9 by gut epithelium was restricted to immature enterocytes of the crypts and the bases of the villi. Therefore, CD9 may be regulated during epithelial differentiation and its function may be limited to interactions with specific extracellular matrix components in the crypt microenvironment. In contrast to PETA-3 and CD9, CD63 expression was localized to granules or structures located in the apical cytoplasm, below the brush border. Ultrastructural studies would be necessary to determine whether CD63 is expressed on the brush border membrane. CD63 has been shown to possess a lysosomal targeting sequence (Metzelaar et al. 1991) and was demonstrated to localize in Weibel-Palade bodies of endothelial cells and platelet granules (Vischer and Wagner 1993; Nieuwenhuis et al. 1987). The apical region of the enterocyte cytoplasm is rich in multivesicular bodies that express major histocompatibility complex (MHC) Class II proteins (Mayrhofer and Spargo 1990). Therefore, CD63 may be expressed in the early endocytic pathway in enterocytes. Furthermore, other members of the TM4SF, CD37, CD53, CD81 (TAPA-1) and CD82 (R2/C3) have been shown to associate with MHC Class II molecules (Angelisova et al. 1994; Schick and Levy 1993), suggesting that CD63 is also involved in these complexes.

Simple epithelia in several other tissues also expressed very high levels of PETA-3. Such structures included bile ducts, terminal bronchioles, and pancreatic intralobular ducts. CD9, CD63, and β1 integrins showed similar localization in these tissues, although CD9 was not detected on pancreatic ducts. Interestingly, staining of bile duct and pancreatic duct epithelia for these antigens was diffuse, suggesting cytoplasmic localization. However, studies on biopsy material would be required to eliminate postmortem degeneration as a cause for this appearance.

Previous studies with MAb 14A2.H1 have characterized the expression of PETA-3 in hemopoietic cells within bone marrow and peripheral blood. These studies showed expression to be restricted to megakaryocytes and platelets (Ashman et al. 1991; von dem Borne 1989). In tonsil sections, PETA-3 expression was reported on the squamous epithelium and blood vessels (Ashman et al. 1991). The results of the present study are in general agreement with these previous findings on hemopoietic cells and tonsil. However, some immature cell types within the BMMNC population and follicular dendritic cells within tonsil follicles also showed PETA-3 expression. The most likely cause of these differences may be the use of MAb 14A2.H1 in the earlier study. 14A2.H1 displays weaker staining of the PETA-3 antigen than MAb 11B1.G4, which was used in the present investigation. Furthermore, the recognition of PETA-3 by 14A2.H1 has been demonstrated to be fixation- and glycosylation-sensitive. In the present study methanol rather than buffered formol acetone was used as fixative. Lymphocytes in blood and in organized secondary lymphoid tissues (spleen and tonsil) did not express PETA-3 or the other tetraspans investigated. An interesting exception was the population of IELs in the small intestine. These cells, which appear to be extrathymically-derived T-cells, express several unique antigens, including the αEβ7 integrin (Yuan et al. 1991). This appears to be the first report describing expression of tetraspan molecules CD9 and CD63 by IELs, and their restriction to this subset of lymphocytes may be related to the specialized microenvironment occupied by these cells.

The tissue distribution of CD9, as described in the present study, is in general agreement with previous work (Rossler et al. 1992; Bouchiex et al. 1985; Jones et al. 1982), with the exception of skeletal and cardiac muscle. Skeletal muscle had been reported as being negative and cardiac muscle positive by anti-CD9 MAb ALB₆ or DU-ALL-1. The opposite was observed in this study. Such discrepancies may arise because different anti-CD9 MAbs may not necessarily be equivalent in their recognition of CD9. For example, the glycosylation state of CD9 in different cell types may vary, thus modifying antigen recognition by certain antibodies.

In summary, this study has shown that PETA-3 co-localizes with β1 integrins and the other tetraspans CD9 and CD63 in particular tissues. However, it is important to note that distinct localization of several antigens investigated occurred in several tissues. This may indicate that tetraspan molecules share common functions in a number of tissues but that they may also have specific functions in some cell types. This is exemplified by PETA-3 in cardiac muscle, CD9 in tissue components of the brain and medullary rays of the kidney, CD63 in pancreatic ancinar cells, and the apical localization of CD63 in enterocytes. Furthermore, co-expression of these antigens by a given cell type may not always imply that they form complexes. For example, compartmentalization of CD63 within intracellular granules in enterocytes would prevent its association with surface antigens. Whether the complexes formed by tetraspans and integrins are constitutive or inducible remains to be investigated. However, the co-localization of these antigens in normal tissues provides a basis for such interactions to occur.

Footnotes

Acknowledgements

We thank Kathy Cash for assistance with cutting the tissue sections, Antony Cambareri and Ly Nguyen for producing the 11B1.G4 antibody, and Stephen Fitter for valuable discussion.

PMS is a postgraduate scholar of the National Heart Foundation of Australia. LKA is a Senior Research Fellow of the National Health and Medical Research Council of Australia.

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