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(Journal of Leukocyte Biology. 2001;69:944-950.)
© 2001 by Society for Leukocyte Biology

Tissue distribution of CD6 and CD6 ligand in cattle: expression of the CD6 ligand (CD166) in the autonomic nervous system of cattle and the human

A. Konno*, J.-S. Ahn{dagger}, H. Kitamura*, M. J. Hamilton{dagger}, J. A. Gebe{ddagger}, A. Aruffo{ddagger} and W. C. Davis{dagger}

* Department of Biomedical Sciences, Graduate School of Veterinary Medicine, Hokkaido University, Sapporo, Japan;
{dagger} Department of Veterinary Microbiology and Pathology, Washington State University, Pullman, Washington; and
{ddagger} Bristol-Myers Squibb Pharmaceutical Research Institute, Princeton, New Jersey

Correspondence: Akihiro Konno, Department of Biomedical Sciences, Graduate School of Veterinary Medicine, Hokkaido University, Sapporo 060-0818, Japan. E-mail: akonno{at}vetmed.hokudai.ac.jp


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ABSTRACT
 
We studied the tissue distribution of CD6+ lymphocytes and cells expressing the CD6 ligand (also known as activated leukocyte cell adhesion molecule CD166) in calves by immunohistochemistry using an anti-bovine CD6 monoclonal antibody (mAb), a human CD6 (huCD6)-immunoglobulin G1 fusion protein (huCD6-Ig), and an anti-human CD166 (anti-huCD166) mAb. The huCD6-Ig and anti-huCD166 mAb bound to the sympathetic and parasympathetic nerve fibers but not to myelinated nerve fibers in the spinal nerve. Studies with human tissue using the anti-huCD166 mAb yielded identical patterns of labeling. Dense accumulations of CD6+ lymphocytes were present in areas of the thymuses and spleens of calves, in areas innervated by huCD6-Ig+ nerves. The cDNAs encoding the bovine CD166 and CD6 were isolated from the sympathetic ganglion and spleen, respectively. Predicted amino acid residues that are important for human and mouse CD6-CD166 binding were also conserved in bovine CD6 and CD166. Bovine CD166 transcripts were detected by reverse transcriptase-PCR in all the tissues that bound huCD6-Ig. These results show that the bovine orthologue of CD166 was constitutively expressed in the autonomic nervous systems of cattle and suggest that CD6+ lymphocytes adhere to CD166+ autonomic nerve terminals via CD6.

Key Words: bovine • {gamma}{delta} T cell • adhesion molecule • sympathetic nerve • parasympathetic nerve


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INTRODUCTION
 
Human CD6 (huCD6) is a 130-kDa glycoprotein expressed on the surface of thymocytes, mature T cells, and a small subset of B cells known as B-1 cells [1 ]. CD6 is a member of the scavenger receptor cysteine-rich super family, which includes the type I macrophage scavenger receptor and the leukocyte antigens CD5, CD163, and bovine WC1 [1 ]. The WC1 molecule is expressed on a major subset of peripheral blood {gamma}{delta}T cells in cattle [2 3 4 ]. The ligand for human and mouse CD6 (CD166) is also called the activated leukocyte cell adhesion molecule. Analysis of the expression of CD166 by activated peripheral blood mononuclear cells has indicated that its expression by mitogen-activated peripheral blood T cells peaks 72 h after stimulation and returns to undetectable levels between days 5 and 8 [5 ]. Initial studies on the role of CD6-CD166 interactions have shown that this receptor-ligand pair is able to mediate the adhesion of CD6-expressing cells to thymic epithelial cells in vitro [6 ]. The data suggest that these molecules could mediate important interactions during T-cell development in the thymus. In the nervous system of chickens, the orthologue of CD166, BEN, has been shown to mediate heterophilic interactions with the neuron-glial cell adhesion molecule and other uncharacterized proteins [7 ].

Herein, we describe the distribution of CD166 in bovine and human tissues. We show that CD166 was also expressed in both the sympathetic and parasympathetic divisions of the autonomic nervous system. In addition, immunohistochemical analysis of the distribution of CD6+ lymphocytes suggested that CD6+ lymphocytes may interact with CD166+ autonomic nerves in vivo.


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MATERIALS AND METHODS
 
Animals and tissue preparation for immunohistochemistry
The tissues used in this study were obtained during necropsy from seven Holstein-Friesian calves (2 days to 6 months old) that were healthy at the time of sacrifice. Tissue samples were collected from the following regions: (1) the central nervous system, including parietal lobes of the cerebrum, the medulla oblongata, and thoracic segments of the spinal cord; (2) the peripheral nervous system, including the seventh cervical nerve (spinal nerve), the cervicothoracic ganglion (sympathetic division of the autonomic nervous system [ANS]), and the dorsal vagus nerve trunk (parasympathetic division of ANS); (3) the endocrine system, particularly the adrenal gland; (4) lymphoid organs, including the thymus, popliteal lymph node, and spleen; and (5) other tissues, including the caudal lobe of the lung, ileum, liver, kidney, and cervical skin. In addition the superior cervical ganglion (sympathetic division of ANS), recurrent laryngeal nerve (parasympathetic division of ANS) that is a branch of the vagus nerve, and sciatic (spinal) nerve were collected from a 70-year-old man who died of mycotic pneumonia. The tissues were embedded in OCT compound (Miles, Elkhart, IN) and then snap frozen in liquid nitrogen.

Immunohistochemistry
Cryostat sections 5 to 6 µm thick were prepared and mounted onto 4% organosilane (3-aminopropyl triethoxysilane; Sigma, St. Louis, MO)-coated glass slides. The sections were fixed in absolute ethyl alcohol at 4°C for 8 min. After rinsing with phosphate-buffered saline (PBS), the sections were blocked with 10% normal goat serum at room temperature for 45 min. Bovine tissue sections were incubated with a fusion protein containing huCD6 and human immunoglobulin (Ig) G1 (huCD6-Ig) [8 ] diluted with PBS at 10 µg/mL, anti-human CD166 (-huCD166) monoclonal antibody (mAb), 3A6 (IgG1) (Ancell, Bayport, MN) at 10µg/mL, or anti-bovine CD6 (-boCD6) mAb, BAQ91A (IgG1) [9 ] at 20 µg/mL for 2 h. After rinsing with PBS, the sections were incubated with biotin-conjugated goat anti-human IgG antibody (Vector, Burlingame, CA) (1:150 in PBS) or anti-mouse IgG1 antibody (Vector) (1:100 in PBS) for 1 h at room temperature. Thereafter, the sections were steeped in 0.9% H2O2 in methanol to inactivate endogenous peroxidase activity at 4°C overnight. After further rinsing with PBS, the sections were incubated with avidin-biotin-peroxidase complex (Vector) for 30 min. After rinsing with PBS, the sections were developed with diaminobenzidine containing 10 mM sodium azide and counterstained with methyl green. Another fusion protein comprising the three outermost domains of bovine WC1 and human IgG1 (WC1D1-3-Ig) (10 µg/mL in PBS) or normal mouse serum (1:200 in PBS) was used as a negative control. Previous studies established that WC1D1-3-Ig does not bind to any cells [J.-S. Ahn, A. Konno, M. J. Hamilton, J. A. Gebe, and W. C. Davis, unpublished results].

Molecular cloning and gene structures of the bovine activated leukocyte cell adhesion molecule (CD166) and CD6 orthologues
A cervicothoracic ganglion was taken from a calf and kept in liquid nitrogen until RNA extraction. Total RNA was isolated from the tissue using Isogen (Nippon Gene, Toyama, Japan) according to the manufacturer’s instructions. The first-strand cDNA was synthesized from the isolated RNA using Oligo(dt)12-18 primer (Life Technologies, Gaithersburg, MD) and superscript reverse transcriptase (RT) (Life Technologies). The cDNA was used to clone the bovine CD166 (boCD166) gene. A set of primers based on the conserved sequences of human and mouse CD166 was designed to amplify the gene. Initial cloning with the PCR was performed with primers (sense primer 5'-ATC ACA TGG TAC AGG AAT GG-3', antisense primer 5'-GGA GTT TAC TGT TCT CTC CA-3') and the PCR reagent system (Life Technologies). For PCR, the ganglion cDNA was denatured at 95°C for 1.5 min, annealed at 54°C for 1 min, and extended at 72°C for 1 min for 30 cycles using the Gene Amp PCR system 2400 (Perkin-Elmer, Norwalk, CN). The PCR product was ligated into a pCR 2.1 vector (Invitrogen, Carlsbad, CA), cloned, and then sequenced using a Dye Terminator Cycle sequencing kit (Perkin-Elmer) according to the manufacturer’s instructions. To obtain 5' and 3' flanking sequences, further PCR amplifications were performed using two sets of primers. The sequences of the set of primers used to obtain the 5' flanking sequence were 5'-GCCCACCAAGAAGGAGGAGG-3' (sense primer) and 5'-CCG CTC CTT CAA CAG CTT GC-3' (antisense primer). The set of the primers used to obtain the 3' flanking sequence were 5'-CTT GCA CAG CAG AAA ACC AG-3' (sense primer) and 5'-ACA ATC CAC GTT CAT GCT TC -3' (antisense primer). The antisense primer used to amplify the 5' flanking sequence and the sense primer used to amplify the 3' flanking sequence were designed from the partial sequence obtained from the initial clone of boCD166. The design of the sense primer used to amplify the 5' flanking sequence and the antisense primer used to amplify the 3' flanking sequence was based on a sequence obtained from human CD166 (huCD166) cDNA. The PCR products were also ligated into the vector, cloned, and sequenced as described above. To obtain the full sequence of boCD166 gene open reading frame with high fidelity, the ganglion cDNA was amplified using the Advantage-HF PCR kit (CLONTECH, Palo Alto, CA). A set of primers (sense primer 5'-CTC ACT AGT ATG GCT TCG AAG GCG GCC CCC-3', anti-sense primer 5'-CTC GGA TCC TTA GGC TTC TGT TTT GTG ATT GTT TTC TTC-3'), based on the draft sequence of the boCD166 gene, was used for the PCR. The PCR product was treated with SpeI and BamHI restriction enzymes (Takara, Tokyo, Japan), ligated into Bluescript SK+ vector (Toyobo, Tokyo, Japan) and then cloned. Four clones were sequenced as described above.

To study the sequence of the boCD6 gene, spleen cDNA was synthesized from calf spleen total RNA as described above. A set of primers based on conserved sequences from human and mouse CD6 was designed to clone boCD6. Cloning by PCR was performed using primers (sense primer 5'-GCT CAG AGC ACC AGT-3', antisense primer 5'-AGG AGG AGA ATT CCC AG-3'). For PCR, the spleen cDNA was denatured at 95°C for 1.5 min, annealed at 52°C for 1 min, and extended at 72°C for 1 min for 30 cycles. The PCR product was ligated into the pCR 2.1 vector and cloned. Six clones were sequenced as described above.

Sequence comparisons were made using the FASTA algorithm (National Institute of Genetics, Mishima, Japan). BoCD166 and CD6 sequences were submitted to the DNA Data Bank of Japan (accession nos. AB039957 and AB042274, respectively; National Institute of Genetics).

RT-PCR study of boCD166 expression
Total RNA was obtained from the cerebral cortex, medulla oblongata, thoracic spinal cord, cervicothoracic ganglion (sympathetic ganglion), adrenal gland, thymus, spleen, and popliteal lymph node from two calves. cDNA was synthesized from the total RNA as described above. PCR studies were performed with the boCD166-specific primers (sense primer 5'-AAT GGT AAC CCT CCT CCT GA-3', antisense primer 5'-CAC AGA CAT AGT TTC CAG CA-3') designed in the present study. For PCR, the cDNA was denatured at 95°C for 1.5 min, annealed at 54°C for 1 min, and extended at 72°C for 1 min for 30 cycles. Bovine ß-actin-specific primers (sense primer 5'-ACC AAC TGG GAC CAC ATG GAG-3', antisense primer 5'-GCA TTT GCG GTG GAC AAT GGA-3') were used as a positive control for RT-PCR [10 ].


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RESULTS
 
Immunohistochemistry
Distribution of huCD6-Ig-binding cells in neuroendocrine system of cattle
huCD6-Ig bound to cell membranes of sympathetic neurons and unmyelinated nerve fibers of the cervicothoracic ganglion and unmyelinated nerve fibers of the dorsal vagus nerve trunk of cattle (Fig. 1a and b ). huCD6-Ig bound to unmyelinated nerve fibers in the spinal nerves—probably efferent secondary fibers of the autonomic pathway going to the skin (Fig. 1c) . huCD6-Ig did not bind to myelinated nerve fibers that comprise the somatic sensory and motor nervous systems (Fig. 1c) . In the thoracic spinal cord of calves, dense aggregations of huCD6-Ig-binding nerve cell bodies and nerve fibers were present in the dorsal commissure extending to the intermediolateral column of each side, where efferent primary neurons of the sympathetic pathway are located [11 , 12 ]. Although aggregations of huCD6-Ig-binding nerve cell bodies and their nerve fibers were seen in the medulla oblongata, which may include parasympathetic nuclei, precise identification of the nuclei was difficult. In the cerebral cortex as well as the medulla oblongata and spinal cord, the pia mater was positive for CD166. Neurons and glial cells of the cerebral cortex were negative. In endocrine tissues, huCD6-Ig bound to cell membranes of adrenal chromaffin cells. These cells develop from migrant nerve cells, which are the homologues of sympathetic efferent neurons (Table 1 ). Negative-control fusion protein WC1d1-3-Ig did not bind to any structures of the neuroendocrine tissues (Fig. 1d 1e 1f) .



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  		Figure 1. Immunoperoxidase-stained sections of bovine peripheral nervous tissues with huCD6-Ig. huCD6-Ig binds to cell membrane of neurons, satellite cells and unmyelinated nerve fibers in the cervicothoracic ganglion (sympathetic ganglion) (a) and unmyelinated nerve fibers in the vagus nerve (parasympathetic nerve) (b). Although huCD6-Ig binds to unmyelinated nerve fibers (autonomic nerve fibers) (arrowheads) in the seventh cervical nerve (spinal nerve), myelinated nerve fibers are negative for CD6 ligand (c). Negative-control fusion protein WC1D1-3-Ig did not bind to any structures in the sympathetic ganglion (d), vagus nerve (e), and spinal nerve (f). Magnification: a–c: x150; d–f: x135.


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  		Table 1. Reactivity of huCD6-Ig in Sections of Normal Calf Tissues

huCD166 immunoreactivity in peripheral nervous systems of cattle and the human
Anti-huCD166 mAb positively stained the cell membranes of sympathetic neurons and their nerve fibers in the sympathetic ganglia, unmyelinated nerve fibers in the vagus, or recurrent laryngeal nerve and unmyelinated nerve fibers in the spinal nerves of cattle (Fig. 2a 2b 2c ) and the human (Fig. 2d 2e 2f) . Myelinated nerve fibers in the spinal nerves of cattle and the human were negative for the antigen (Fig. 2c and 2f) .



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  		Figure 2. Immunoperoxidase-stained sections of calf and human peripheral nervous tissues with anti-huCD166 mAb. The calf (a–c) and human (d–f) nervous tissues stained with the mAb showed identical labeling to the sections stained with huCD6-Ig (Fig. 1a 1b 1c) . Panels a–c: calf cervicothoracic ganglion (a), vagus nerve (b), and seventh cervical nerve (c). Panels d–f: human superior cervical ganglion (sympathetic ganglion) (d), recurrent laryngeal nerve (parasympathetic nerve) (e), and sciatic nerve (spinal nerve) (f). Arrowheads point to CD166-immunoreactive unmyelinated nerve fibers in the spinal nerves (2c and f). Magnification, x125.

Distribution of huCD6-Ig-binding cells in non-neuroendocrine tissues of cattle
huCD6-Ig bound to nerve fibers and nerve plexus in all the tissues examined. These nerve fibers innervated arterial and arteriolar walls. In addition these fibers innervated, the thymic medulla (Fig. 3a and c ), periarterial lymphatic sheath (PALS) (Fig. 4a and b ) and red pulp of the spleen (Fig. 4a and 4c) , lamina propria of the ileum, and dermis of the skin. huCD6-Ig also bound to epithelial cells in the cortex and medulla of the thymus (Fig. 3a 3b 3c) , in the crypt of the ileum, in the bile duct of the liver, in the bronchi and bronchioli of the lung, and in the hair follicles and sebaceous and sweat glands of the skin. In addition huCD6-Ig bound to sinus macrophages in the lymph node (Fig. 5a and b ). Mononuclear cells scattered throughout the red pulp of the spleen were labeled also (Table 1) . Negative control fusion protein WC1d1-3-Ig did not bind to any cells in the thymus (Fig. 3d) , spleen (Fig. 4d) , lymph node (Fig. 5c) , and other tissues examined in this study.



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  		Figure 3. Immunoperoxidase-stained sections of calf thymus with huCD6-Ig (a–c) and anti-CD6 (e–g). Panels: middle-power view of a section stained with huCD6-Ig (a; many huCD6-binding cells are located in the medulla); high-power view of the cortex (b; huCD6-Ig binds to fine cytoplasmic processes of epithelial cells in the cortex); high-power view of the medulla [c; nerve fibers (arrowheads) and epithelial cells (arrows) bind the fusion protein]; negative-control section stained with WC1-Ig shows no immunoreactivity (d); middle-power view of a section stained with anti-CD6 mAb (e; CD6+ lymphocytes are rare in the cortex but densely distributed in the medulla); high-power view of the cortex (f; epithelial cells show a weak positive reaction for CD6); high power of the medulla (g; most lymphocytes express CD6); negative-control section stained with mouse IgG shows no immunoreactivity (h). C, cortex; M, medulla. Magnifications: panels a, d, e, and h, x72; panels b and f, x320; panels c and g, x200.



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  		Figure 4. Immunoperoxidase-stained sections of calf spleen with huCD6-Ig (a–c) and anti-CD6 mAb (e–g). Panels: middle-power view of a section stained with huCD6-Ig [a; filamentous structures labeled with huCD6-Ig are seen in the periarterial lymphatic sheath (PALS) of white pulp and in the red pulp]; high-power view of the PALS (b; huCD6-Ig-binding nerve fibers innervate the PALS around central arteries); high-power view of the red pulp [c; fine nerve fibers labeled with huCD6-Ig (arrowheads) are seen throughout the red pulp]; negative-control section stained with WC1-Ig shows no immunoreactivity (d); middle-power view of a section stained with anti-CD6 mAb (e; CD6+ lymphocytes aggregate in the PALS and red pulp); high-power view of the PALS (f; CD6+ lymphocytes are clustered densely in the PALS); high-power view of the red pulp (g; in the red pulp, CD6+ lymphocytes constitute a considerable proportion of the cells of this area); negative-control section stained with mouse IgG shows no immunoreactivity (h). C, central artery; WP, white pulp; RP, red pulp; mz, marginal zone. Magnifications: panels a, d, e, and h, x80; panels b, c, f, and g, x180.



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  		Figure 5. Immunoperoxidase-stained sections of calf lymph node with huCD6-Ig (a and b) and anti-CD6 mAb (d and e). Low-power view of a section stained with huCD6-Ig (a; a large number of cells labeled with huCD6-Ig were located in the medulla); high-power view of the medulla (b; in the medulla, huCD6-Ig bound to macrophages filling the sinus); a negative-control section stained with WC1-Ig showed no immunoreactivity (c); low-power view of a section stained with anti-CD6 mAb (d; CD6+ lymphocytes were seen in the interfollicular space and paracortex); high-power view of the medulla. CD6+ lymphocytes scattered in the medullary cords (e; a small number of the CD6+ cells were also seen in the sinus); negative-control section stained with mouse IgG showed no immunoreactivity (f). F, lymphoid follicle; P, interfollicular space and paracortex; M, medulla; MC, medullary cord; S, sinus. Magnifications: panels a, c, d, and f, x25; panels b and e, x200.

Distribution of boCD6+ cells:
In the thymus, CD6+ thymocytes were sparse in the cortex, and densely distributed throughout the medulla (Fig. 3e 3f 3g) . Cortical epithelial cells showed a weak positive reaction with anti-CD6 mAb (Fig. 3f) . In the spleen, CD6+ lymphocytes were aggregated in the PALS and red pulp (Fig. 4e 4f 4g) . In the lymph node, CD6+ lymphocytes were seen in the interfollicular space and paracortex (Fig. 5d) . CD6+ lymphocytes were also seen in the medullary cords and medullary sinus (Fig. 5e) . In the skin, CD6+ lymphocytes were scattered in the dermis, particularly the deep (reticular) dermis. In the ileum, CD6+ intraepithelial lymphocytes were located from the bottom to the tip of the villus. CD6+ cells were rarely seen in the liver, kidney, and adrenal gland. Negative-control mouse IgG did not stain any cells in the thymus (Fig. 3h) , spleen (Fig. 4h) , lymph node (Fig. 5f) , and other tissues examined.

Cloning and characterization of the boCD166 and boCD6 orthologues
The cDNA encoding boCD166 was cloned from a calf sympathetic ganglion. The cloned boCD166 cDNA sequence contained 1,749 base pairs (bp) in the open reading frame. boCD166 cDNA showed homologies of approximately 93 and 89% with huCD166 and mouse CD166 cDNA, respectively, which were lower than the predicted amino acid sequence homologies of approximately 95 and 92%, respectively. Nine predicted amino acid residues of extracellular domain 1 that are important for huCD166 and mouse CD166 binding to CD6 [13 ] were conserved in cattle (Fig. 6 ).



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  		Figure 6. Alignment of huCD166 and mouse CD166 (moCD166) domain 1, CD6-binding-domain amino acid sequences with predicted boCD166 domain-1 amino acid sequence (A). Residues in huCD166 domain 1 whose mutation affected CD6 binding are boxed [5 ]. Alignment of hu- and moCD6 domain 3, CD166-binding domain, and amino acid sequences with predicted boCD6 domain-3 amino acid sequence (B). Residues in huCD6 domain 3 whose mutation affected CD166 binding are boxed [1 ]. Residues whose mutation significantly compromised huCD6 expression and/or structure are underlined [1 ]. Residue Asn345 in huCD6 was a potential N-linked glycosylation state [1 ].

The cDNA encoding boCD6 was also cloned from calf spleen cDNA. The boCD6 cDNA sequence contained 429 bp, which probably encodes the extracellular-domain-3, stalk region, and transmembrane segment. boCD6 cDNA showed homologies of approximately 86 and 75% with huCD6 and mouse CD6 cDNA, respectively, which was higher than the predicted homologies of 76 and 69%, respectively. Predicted amino acid residues in huCD6 and mouse CD6 domain 3, involved in binding to CD166 [1 ], were conserved in cattle (Fig. 6) . Residue Asn345 in human and mouse CD6, which is a potential N-linked glycosylation site, was also conserved in cattle. Of 10 residues whose mutation significantly compromised human and mouse CD6 expression and/or structure, 9 were conserved in cattle.

Expression and distribution of boCD166 mRNA
As shown in Figure 7 , a transcript (361 bp) for boCD166 was detected in all tissues examined. A transcript (890 bp) of bovine ß-actin as a positive control for RT-PCR was also detected in all the tissues, whereas no signal for ß-actin was detected in RT-negative PCR (data not shown).



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  		Figure 7. RT-PCR analysis of boCD166 mRNA expression in calf tissues that contain huCD6-Ig-binding cells. Five nanograms of cDNA were subjected to PCR using specific primers for boCD166 or ß-actin as positive controls for RT-PCR. The PCR products were separated on 2% agarose gels and visualized by ethidium bromide staining. Bovine CD166 (361 bp) (top) and ß-actin specific transcripts (890 bp) (bottom) were detected in all the tissues.


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DISCUSSION
 
In humans and mice, the role of CD6 as an accessory molecule in T-cell activation is clearly established. Experiments with anti-CD6 and anti-CD3 mAbs indicate that CD6 is a signaling molecule that modulates T-cell receptor signaling [14 , 15 ]. Moreover, CD166 mRNA is detected in activated monocytes, suggesting that the interaction of CD6 with CD166 on antigen-presenting cells may play an important role in activating T cells during the primary immune response [1 , 5 ]. The present study showed that huCD6-Ig binds to sinus macrophages in the lymph node. The sinus macrophages in lymph nodes are large and contain large amounts of cytoplasm. These macrophages are thought to be activated cells. Wee et al. [8 ] have reported the binding of huCD6-Ig to mononuclear cells in the sinus of the mouse lymph node. These cells were probably sinus macrophages. In the lymph nodes of calves, CD6+ lymphocytes were scattered around the sinus macrophages, suggesting an interaction between CD6+ lymphocytes and CD166+ macrophages.

In the present study, huCD6-Ig also bound to hepatocytes and epithelial cells, including thymic epithelial cells. These observations were consistent with those obtained with an anti-CD166 mAb, J4-81, in human tissue [6 ], indicating that the huCD6-Ig used in this study detected boCD166. huCD6 and mouse CD6 bind to CD166 expressed on thymic epithelial cells [6 , 8 ]. In the thymus, CD6 and CD166 have been thought to mediate interactions between thymocytes and thymic epithelial cells [6 , 8 ]. The bovine thymic epithelial cells expressed not only the CD6 ligand, but also CD6. Binding of CD6-CD166 may be associated with meshwork formation of epithelial cells in the bovine thymus.

In humans and mice, CD6 has been detected in neurons randomly scattered in the parenchyma of all brain areas [16 ]. huCD166 and the rat orthologue, KG-CAM, have also been detected in neurons of the cerebral cortex [6 , 17 ]. However, the anti-boCD6 mAb and huCD6-Ig did not bind to neurons or glial cells in the parenchymal tissue of calf cerebral cortex. The data presented here show that huCD6-Ig reacts with sympathetic and parasympathetic neurons and their nerve fibers in the central (medulla oblongata and spinal cord) and peripheral nervous tissues in calves.

In the present study, positive immunoreactivity with anti-CD166 mAb as well as huCD6-Ig was seen in the autonomic nervous systems of cattle. We also demonstrated that boCD166 mRNA was expressed in the autonomic nervous system. In addition CD166 immunoreactivity in the human tissues assured that the molecule detected in bovine tissues by huCD6-Ig and anti-CD166 mAb was the boCD166 orthologue. This is the first report of expression of CD166 in peripheral autonomic nervous tissues of mammals. Although expression of CD166 on autonomic neurons has not been reported in mammals, BEN, which is the chicken orthologue for CD166, has been detected in nervous tissue, including autonomic neurons [19 20 21 ]. The studies of BEN support our contention that CD166 is expressed on autonomic neurons of cattle and humans. The protein known as BEN is recognized by an mAb (anti-BEN mAb) that was originally generated in mice against surface determinants of the epithelial component of the bursa of Fabricius [19 ]. In the embryonic nervous system of chickens, BEN is expressed on several types of neurons whose axons project to the periphery. These include the motor neurons of the brain and spinal cord, the sensory neurons of the dorsal root ganglion, the cranial ganglia, the sympathetic ganglia, and the neurons of the enteric nervous system [19 20 21 ]. The distribution of BEN in the nervous system is broad. However, in humans and cattle, CD166 and its bovine orthologue were not expressed on myelinated nerve fibers, indicating that expression of CD166 in the nervous system is restricted to autonomic nerves in the human and cattle. The expression of BEN in the nervous tissues, except for enteric plexus, is transient during embryogenesis; however, CD166 immunoreactivity was detected in bovine and human nervous systems even at 6 months and 70 years of age, respectively.

Autonomic nerve fibers are unmyelinated and it is difficult to distinguish axons from Schwann cells in peripheral tissue at the light-microscopic level. However, the fusion protein and mAb bound to cell membranes of neurons in the sympathetic ganglion and nerve fibers in the spinal cord and medulla oblongata, which contain no Schwann cells. In addition, Schwann cells surrounding axons in myelinated nerve fibers were negative for CD166. These results indicate that the fusion protein binds to axons alone within the autonomic nerve fibers, suggesting that CD166 is expressed on nerve terminals. Electron-microscopic studies revealed synaptic contact of sympathetic nerve terminals with splenic T lymphocytes [18 ]. CD6-CD166 binding may be involved in this contact.

CD6+ lymphocytes were concentrated in the thymic medulla, PALS, and red pulp of the spleen and were sparsely scattered throughout the dermis, which contained innervation of huCD6-Ig-binding nerves, suggesting the binding of CD6+ lymphocytes to CD166+ nerves in vivo. In cattle, the population of CD6+ lymphocytes contains {alpha}ß and WC1- {gamma}{delta} T cells. These are present in the thymic medulla, paracortex of the lymph nodes, and dermis [22 , 23 ]. In the spleen, WC1- {gamma}{delta} T cells are primarily concentrated in the red pulp, whereas {alpha}ß T cells are primarily concentrated in the PALS [23 ]. Study of the interaction between autonomic nerves and CD6+ lymphocytes, particularly WC1- {gamma}{delta} T cells of the splenic red pulp, may help elucidate the role of WC1- {gamma}{delta} T cells in homeostasis and host defense.


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ACKNOWLEDGEMENTS
 
This study was partially supported by a Grant-in-Aid for Scientific Research from the Ministry of Education, Science, Sports and Culture, Japan (no. 11760199), USDA-NRICGP (no. 98-02480), the College of Veterinary Medicine Animal Health Research Center intramural grant (WNV-00138), and the Washington State Monoclonal Antibody Center.

The authors thank Dr. S. Ritter, Washington State University College of Veterinary Medicine, for her valuable suggestions on this work. We also thank Dr. K. Nagashima and H. Sawa, Hokkaido University Graduate School of Medicine, for their kind assistance in the collection of human nervous tissues.

Received June 12, 2000; revised December 1, 2000; accepted January 22, 2001.


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K. Thelen, T. Georg, S. Bertuch, P. Zelina, and G. E. Pollerberg
Ubiquitination and Endocytosis of Cell Adhesion Molecule DM-GRASP Regulate Its Cell Surface Presence and Affect Its Role for Axon Navigation
J. Biol. Chem., November 21, 2008; 283(47): 32792 - 32801.
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