A novel molecular interaction for the adhesion of follicular CD4 T cells to follicular dendritic cells

Abstract

Nectins and Nectin-like molecules (Necls) play a critical role in cell polarity within epithelia and in the nervous and reproductive systems. Recently, immune receptors specific for Nectins/Necls have been described. Since expression and distribution of Nectins/Necls is often subverted during tumorigenesis, it has been suggested that the immune system may use these receptors to recognize and eliminate tumors. Here we describe a novel immunoreceptor, WUCAM, which is expressed on human follicular B helper T cells (Tfh) and binds a Nectin/Necl family member, the poliovirus receptor (PVR), under both static and flow conditions. Futhermore, we demonstrate that PVR is abundantly expressed by follicular dendritic cells (FDC) within the germinal center. These results reveal a novel molecular interaction that mediates adhesion of Tfh to FDC and provide the first evidence that immune receptors for Nectins/Necls may be involved the generation of T cell-dependent antibody responses.

Introduction

Nectins and nectin-like (Necl) molecules are immunoglobulin-like cell adhesion molecules that maintain polarity and organization of epithelium, neuronal and germinal cells [1, 2]. Some of these molecules also function as receptors for poliovirus and herpes simplex viruses [3–5]. To mediate cell-cell junctions, Nectins and Necls trans-interact homophilically and/or heterophilically with specific family members [1, 2]. Moreover, Nectins and Necls cooperate with E-cadherins, integrin α_vβ₃ and growth factor receptors [1, 2]. Normal expression and distribution of Nectins and Necls are crucial for maintaining cell polarity in epithelia, preventing the formation of tumors and metastases [6]. Human non small-cell lung cancers frequently lack expression of Necl-2 (also called tumor suppressor of lung cancer, TSLC), which acts as tumor suppressor [7]. The poliovirus receptor (PVR, also called Necl-5) and Nectin-1 are abnormally expressed on human cancers and may endow cells with a growth advantage and increased migratory capacity [8–11].

Recent work has shown that Nectins and Necls act as ligands for cell surface receptors expressed on lymphocytes. PVR binds both DNAM-1 (CD226) [12, 13] and Tactile (CD96) [14, 15], which are expressed on NK cells, CD8⁺ T cells and CD4⁺ T cells. Additionally, Nectin 2 binds DNAM-1 [12, 13] and Nectin 1 binds Tactile [15]. Necl-2 binds CRTAM, a receptor expressed on activated NK cells and CD8+ T cells [16–18]. Immune receptors binding Nectins and Necls have been shown to promote NK cell adhesion to and/or killing of tumor targets in vitro [12–14, 17, 19, 20]. Furthermore, DNAM-1 and CRTAM have been shown to induce NK cell-mediated rejection of transplanted tumors in vivo [17, 21]. Thus, it has been hypothesized that these immune receptors may be important for recognition and elimination of cells that have acquired an altered expression or distribution of Nectin/Necls as a result of neoplastic transformation. In addition to tumor surveillance, immune receptors for Nectins and Necls may be involved in other critical steps of an immune response, such as migration. Supporting this, DNAM-1, which is expressed not only on lymphocytes but also on monocytes and DC, facilitates monocyte migration through endothelial cells expressing PVR [22].

In this study, we report the identification of a novel immunoreceptor, called Washington University Cell Adhesion Molecule (WUCAM), which is preferentially expressed on human B helper follicular T cells (Tfh). WUCAM binds with high affinity to a Nectin/Necl family member, the poliovirus receptor (PVR). We also show the expression of PVR on human follicular dendritic cells (FDC) in the germinal centers. Together, these results suggest that WUCAM-PVR interactions may be important in regulating T cell function within the germinal center, contributing to T cell-dependent B cell responses.

Results

WUCAM is a novel member of the Ig-superfamily distantly related to Nectins and Necls

The extracellular domains of Nectins and Necls consist of one membrane distal Ig-V-type domain followed by two membrane-proximal Ig-C-type domains [1, 2]. Previous studies indicated that both homotypic and heterotypic interactions of Nectins and Necls involve a binding interface in the C-C'-C"-D β-strands of the Ig-V type domains [23]. Amino acid alignment of this region from Nectins, Necls and their counter-receptors revealed two groups of putative binding interfaces (Supporting Information Fig. 1). While Necl-1-4 and Necl-2-counterreceptor CRTAM had similar binding interfaces, Nectin-1-4, PVR and PVR counterreceptors DNAM-1 (CD226) and Tactile (CD96) clustered differentially. To further our understanding of the interactions among Nectins and Necls and their counter-receptors in the immune system, we searched the National Center Biotechnology Information (NCBI) cDNA database and found an additional candidate receptor that aligned with Nectin1-4, PVR, DNAM-1 and Tactile in the distal Ig-V-type domain. This putative receptor, that we named Washington University cell adhesion molecule (WUCAM), displays a single Ig-V-type domain, a transmembrane spanning sequence and a short cytoplasmic tail (Supporting Information Fig.1). Despite the similarity of WUCAM's putative binding interface with that of other Nectins (up to 50% in between residues C45 and C108 of WUCAM by pairwise alignment), the overall percentage of identity between WUCAM, Nectins, DNAM-1 and Tactile is below 16% and in fact WUCAM has not been recognized as a member of the nectin family.

The cytoplasmic domain of WUCAM exhibited a putative type I PDZ binding motif [24] (Supporting Information Fig. 1). Similar motifs previously found in Nectin/Necls and their countereceptors were shown to recruit membrane-associated guanylate kinases (MAGUKs) and other cytosolic proteins that regulate cell polarity and adhesion by organizing actin cytoskeleton [1, 2, 6]. Additionally, the WUCAM cytosolic domain contained a proline-rich region, which may recruit SH3-domain containing proteins, and two tyrosines, one of which is part of a canonical tyrosine-based inhibitory motif (ITIM) (Supporting Information Fig. 1). ITIM are known to recruit protein tyrosine phosphatases SHP-1 and SHP-2 that inhibit cell activation [25].

Taken together, these cytosolic motifs suggest that WUCAM may deliver intracellular signals that induce cell adhesion and modulate cell activation.

WUCAM binds PVR

To investigate whether WUCAM interacts homotipically or heterotipically with itself or with other members of the Nectin/Necl family, we performed cell-conjugate assays. WUCAM cDNA was expressed in the murine cell line Baf3 as an N-terminus FLAG-tagged fusion protein (WUCAM/Baf3) (Supporting Information Fig. 2). Other Necl family members such as Necl-1, Necl-3 and Necl-4 were also expressed in Baf3 (Supporting Information Fig. 3). PVR, Necl-2, Nectin-1, Nectin-2, DNAM-1, Tactile and CRTAM had been previously cloned and expressed in the human cell line Daudi or in the mouse cell line P815 [14, 17]. After dye labeling, WUCAM/Baf3 cells were co-incubated at 37°C with another transfectant or mock-transfected cells labeled with a different dye and formation of conjugates was measured by two-color flow cytometry. WUCAM/Baf3 cells formed abundant conjugates with PVR/Daudi, but not with mock-transfected Daudi cells or other transfectants (Fig. 1A and data not shown).

Figure 1.

WUCAM binds to PVR. (A) Baf3 cells transfected with WUCAM (WUCAM/Baf3) form conjugates with Daudi cells transfected with PVR (PVR/Daudi) but not mock-transfected-Baf3 or Daudi cells. One experiment representative of 3 is illustrated. (B) Soluble forms of human PVR (PVR-hFc) and mouse Tage4 (Tage4-hFc) bind WUCAM/Baf3 (grey profiles), but not mock-transfected Baf3 (empty profiles). A soluble form of WUCAM (WUCAM-mFc) binds PVR/Daudi (grey profiles), but not control Daudi cells (empty profiles). Moreover, WUCAMmFc does not bind WUCAM/Baf3 or Baf3 (grey and empty profiles respectively). (C) The anti-WUCAM antibody 4E1.2 (black thin dotted line) and the anti-PVR antibody SKII.4 (grey dotted line) block binding of a soluble form of PVR (PVR-hFc) to WUCAM/Baf3 cells to background levels (black thin line). An isotype control antibody does not affect the binding (black solid line). Experiments in (B) and (C) were performed three times with identical results. (D) Detachment profile of WUCAM-Baf3 cells interacting with surface-immobilized PVR-hFc. The percentage of WUCAM/Baf3 (black squares) or mock transfected-Baf3 (empty circles) bound to PVR-hFc is shown as a function of increasing wall shear stress. Cells were allowed to settle onto the indicated surface-immobilized protein substrates for 10 min prior to the application of flow (0.5 to 16 dyn cm-2). Values represent the mean ± SD for two experiments performed in duplicate.

To corroborate WUCAM-PVR interaction we performed binding assays. WUCAM, Tactile, CRTAM, Necl-1, PVR and Necl-2 were expressed as human or mouse IgG-Fc soluble fusion proteins, and binding of soluble molecules to transfected or mock-transfected cell lines was performed. Fc fusion forms of PVR and its mouse homolog, Tage4, specifically bound WUCAM/Baf3 but not mock-transfected Baf3 cells or other transfectants (Fig. 1B). Moreover, soluble WUCAM (WUCAM-mFc) strongly bound to PVR/Daudi but not to mocktransfected/ Daudi or other transfectants (Fig. 1B and Supporting Information Fig. 3).

We also generated the WUCAM-specific mAb 4E1.2 (Supporting Information Fig. 2) and demonstrated that it significantly reduces PVR-hFc binding to WUCAM/Baf3, whereas an isotype-matched control antibody did not (Fig. 1C). Furthermore, a mAb against PVR (SKII.4) also abrogated the WUCAM-PVR interaction (Fig. 1C). To measure the strength of WUCAMPVR interactions, we evaluated the extent of adhesion of WUCAM/Baf3 cells to substrates coated with PVR-hFc or control hIgG using a laminar flow assay. WUCAM/Baf3 cells bound to PVR-hFc resisted detachment by shear forces up to 16 dyn/cm−2 (Fig. 1D). We conclude that WUCAM binds specifically and robustly to PVR, both under static and flow conditions.

WUCAM is expressed on activated CXCR5⁺CD4⁺ T cells in peripheral blood and Tfh cells in tonsils

Having established that WUCAM binds PVR, we then investigated WUCAM expression pattern on immune cells. We initially cloned WUCAM cDNA from human CD56⁺CD3⁻ NK cells. Furthermore, the UCSC Gene Sorter database indicated that WUCAM mRNA is expressed in CD4⁺ T cells, CD8⁺ T cells, and CD56⁺CD3⁻ NK cells. However, we were not able to detect WUCAM on the surface of resting peripheral blood mononuclear cells (PBMC) from healthy donors with mAb 4E1.2 (data not shown and Fig. 2A). Therefore, we asked whether expression of WUCAM requires cell activation. PBMC were stimulated with PHA and IL-2 for different time intervals. A few WUCAM⁺ CD3 T cells appeared as early as 48h after stimulation. However, a distinct WUCAM+ population was detected only 4 to 5 days later (Fig. 2A and 2B). Most WUCAM⁺ cells were CD4⁺ T cells (5–25% of CD4+ T cells in the individuals tested). Additionally, in the donors examined, a variable proportion of CD8+ T cells and CD56⁺CD3⁻ NK cells also expressed WUCAM upon activation. WUCAM expression declined 8–10 days after PHA/IL-2 stimulation, when cells were returning to a resting state. To characterize the peripheral blood CD4⁺ T cells that express WUCAM upon activation, we stained PHA/IL-2 blasts with several markers. A large proportion of the WUCAM⁺CD4⁺ T cells consistently expressed CXCR5 (Fig. 2C), a chemokine receptor that guides B helper follicular CD4⁺ T cells (Tfh) into the follicles to provide help to antigen-activated B cells [26–28].

Figure 2.

WUCAM is expressed upon cell activation. (A) PBMC stained ex-vivo (upper panels) or after in-vitro stimulation for 6 days with PHA and IL-2 (lower panels). Cells were stained with anti-WUCAM and counterstained with anti-CD3, anti-CD56, anti-CD4 and anti-CD8. One donor representative of 3 donors tested is shown. (B) Time course analysis of WUCAM expression on CD4⁺ T cells, CD8⁺ T cells and CD56⁺CD3⁻ NK cells. Cells were stained as in (A). Bars represent the percentage of WUCAM⁺ cells within the indicated lineage at different time points. One donor representative of 3 donors tested is shown. (C) Most of the CD4⁺ T cells expressing WUCAM also express the chemokine receptor CXCR5 (insert). The percentage of WUCAM⁺ cells is reduced in comparison to panels B and C as staining was performed at day 8 after stimulation, when WUCAM expression begins to decline.

To corroborate that Tfh express WUCAM, we isolated cells from the human tonsils and examined them ex-vivo. WUCAM was expressed on a large proportion of CD4⁺ T cells in the tonsil (30–50% depending on the donor) (Fig. 3A). Within CD4⁺ T cells, WUCAM was mostly confined to the CXCR5^high/ICOS^high subset (Fig. 3B) that contains fully differentiated Tfh [26–29]. WUCAM expression was absent on CXCR5-/ICOS_low cells and negative/low on the CXCR5^int cell population that includes Tfh precursor cells [29]. WUCAM⁺ CD4⁺ T cells, in addition to express high levels of ICOS, a well known marker of Tfh [27–29], also expressed the activation marker CD69 (Fig. 3C). In tonsil, WUCAM was also present on some CD3⁺CD8^int T cells but not on CD56⁺CD3⁻ NK cells (Fig. 3A). Moreover, PVR-hFc bound ICOS^high CD4⁺ T cells and this binding was significantly reduced by the WUCAM-specific 4E1.2 antibody (Fig. 3D), thus confirming that the physiological levels of expression of WUCAM detected on Tfh is sufficient to mediate binding to PVR expressing cells.

Figure 3.

WUCAM expression on purified Tfh cells. (A) Tonsil cells were analyzed ex-vivo for WUCAM expression and counterstained with the lineage markers CD3, CD4, CD8, CD56 and CD19. The vast majority of cells expressing WUCAM are confined within the CD4⁺ T cell subset. However, WUCAM expression was repeatedly observed on some CD3⁺CD8^low/int. (B) WUCAM expression is mostly confined to CXCR5^high/ICOS^high CD4⁺ T cells that correspond to Tfh cells. Although the level of WUCAM and the percentages of WUCAM⁺ CD4⁺ T cells varied in different donors, only CXCR5^high/ICOS^high cells within CD4⁺ T cells expressed WUCAM in every sample analyzed. (C) WUCAM⁺ CD4⁺ T cells express high levels of CD69. One specimen representative of three tonsil specimens analyzed is shown. (D) ICOS^high CD4⁺ T cells from tonsil bind soluble PVR (PVR-hFc) but not a control soluble molecule (ctr-hFc). This binding is significantly reduced by the anti-WUCAM antibody 4E1.2, but not by an irrelevant isotypematched control (ctr-IgG3). One experiment representative of two is shown.

Taken together these results indicate that in some specific lymphocytes subsets WUCAM expression is induced by activation. In tonsils, however, WUCAM is constitutively expressed on a subset of T cells that correspond to Tfh cells.

PVR is constitutively expressed by follicular DC

To define the physiological context in which WUCAM+ Tfh cells encounter cells expressing PVR, we analyzed human tonsils, lymph nodes and spleen by flow cytometry and immunohistochemistry with an anti-PVR antibody. PVR was not detected on germinal center B cells, or any other B cell subset (data not shown). We did not detect PVR expression on peripheral blood B cells stimulated with CD40L in the absence or presence of IL-2, IL-10 and/or IL-4. However, PVR was strongly upregulated on peripheral T cells upon activation (data not shown) and more importantly was abundantly expressed within germinal centers, with a pattern that closely resembled that of follicular dendritic cell (FDC) network (Fig. 4A–4D). Thus, the presence of WUCAM on Tfh cells may facilitate their interaction with PVR⁺ FDC within the germinal center.

Figure 4.

PVR is highly expressed by FDC. Immunohistochemical analysis of human lymph node (A, B, E, F) and spleen (C, D) sections reveals expression of PVR on a vast network of cells within the germinal centers that for location, shape and distribution closely resemble the FDC network (A, B, C and D). (E) PVR is also brightly expressed by some IDC in the T cell area of lymph nodes. Other IDC express lower level of PVR. (F) HEV also express PVR. PVR was detected with mAb SKII.4, followed by a biotinylated anti-Ig secondary antibody and streptavidin-immunoperoxidase (brown) or streptavidine-alkaline phosphatase (red). Magnifications: 400×(b and d), 200× (e and f) and 100× (a and c). Scale bars: 50 microns (b and d), 100 microns (e and f) and 200 microns (a and c).

PVR was also expressed at high level on some cells in the T cell area that, by shape and location, corresponded to interdigitating dendritic cells (IDC) (Fig. 4E). Other IDC within the T cell area expressed lower levels of PVR, suggesting the existence of some heterogeneity within IDC, possibly related to differences in the DC maturation stage. Accordingly, in vitro-culture monocyte-derived DC upregulate PVR expression upon maturation [30]. PVR was also expressed by high endothelium venules (HEV) (Fig. 4F). Because WUCAM is expressed ex vivo on Tfh, it is unlikely that Tfh will bind to PVR on HEV or IDC. However, in these locations, PVR may interact with other counter-receptors such as DNAM-1 and Tactile or may interact with activated WUCAM-expressing Tfh-precursors that recirculate between the blood and the lymphoid tissue.

Discussion

Our study demonstrates that human lymphocytes can express WUCAM, a receptor that similarly to DNAM-1 and Tactile binds the Nectin/Necl family member PVR. While DNAM-1 and Tactile are constitutively present on circulating NK cells, CD8⁺ T cells and CD4⁺ T cells, WUCAM expression on circulating lymphocytes is induced by cell activation. Most of the cells that upregulate WUCAM in-vitro belong to the CD4⁺ T cell lineage and express the chemokine receptor CXCR5 that directs T cells to the germinal center. Moreover, WUCAM is expressed ex vivo on CD4⁺CXCR5^high/ICOS^high Tfh in tonsil tissue. These results indicate that WUCAM may participate in the T cell help to B cell responses. Because we did not detect PVR on resting, activated or germinal center B cells, it is unlikely that WUCAM directly contributes to cognate interaction between antigen-specific CD4+ T cells and antigen-presenting B cells. However, we found high level of PVR expression on the FDC network in the germinal center in tonsil. This observation was consistent with the expression of PVR in FDC of murine intestinal Peyer's patches [31]. Therefore, PVR-WUCAM may facilitate Tfh-FDC contacts and positioning of Tfh within the B cell follicle.

One major mechanism by which Tfh home into B cell follicles involves the expression of CXCR5, which endows Tfh cells with responsiveness to CXCL13 [26–28]. However, it has been suggested that additional factors may guide Tfh homing into follicles [26]. Expression of WUCAM on Tfh may be one of these factors by inducing adhesiveness of Tfh to FDC. A recent study indicated that following antigen-induced expansion, Tfh undergo a contraction phase to generate a pool of memory B-helper T cells which are retained within the antigen-draining lymph nodes for prolonged period of time and are readily available for secondary responses [32]. Because of the capacity of WUCAM to mediate strong adhesion to PVR and the delayedupregulation of WUCAM observed in-vitro, WUCAM may facilitate retention of memory Tfh cells within the lymph node. Consistent with this model, WUCAM+ Tfh in secondary lymphoid organs also express igh levels of CD69, which has been shown to mediate lymphocyte retention by negatively regulating the function of sphingosine 1-phosphate receptor-1 [33]. WUCAMPVR interaction may also enhance Tfh proliferation and cytokine or chemokine production. However, since Tfh obtained ex vivo from tonsil are prone to apoptosis (data not shown), the role of WUCAM-PVR on Tfh function could not be directly assessed in vitro, and will require in vivo experimentation. The involvement of WUCAM in T cell-dependent antibody responses is also supported by a recent study demonstrating a defect in IgG and IgA production to orally ingested antigens in PVR-deficient mice [34].

It remains unclear why lymphocytes require at least 3 distinct receptors for PVR: they may take part to different aspects of the immune response, they may be expressed in different compartments or have different signaling capacities. In any case, to ensure redundancy seems a good strategy to preserve a vital function. PVR, for instance, is strongly over-expressed in many tumors and it confers growing and metastatic potential. Thus having three receptors and modulate their expression level at different times and locations during lymphocyte activation, may guarantee a more effective anti-tumor immune surveillance. Ultimately, however, the full understanding of the distinct contributions of Nectins, Necls and their receptors at various stages of the immune response will require further experimentation in in vivo mouse models.

Materials and methods

cDNA cloning and expression

Sequence similarity searches were performed with NCBI BLAST and the HMMER package. Sequence manipulations were made in the Bioedit program (http://www.mbio.ncsu.edu/BioEdit/bioedit.html). Multiple sequence alignments were made with the T-Coffee program. WUCAM (Washington University cell adhesion molecule, NCBI accession BC101289) was amplified by PCR from human CD56⁺CD3⁻ NK cell cDNA. WUCAM primers for PCR were 5’-CTCAGCCAACAGTTCCTCATCAGTAGC-3’ to 5’-CTCTGGTTGCTAACCAGTCTCTGTG-3’. Tage4 was cloned by PCR using C57BL/6 whole brain cDNA with primers 5’-CCACATGGCCCCACTCGCCGGTG-3’ t o 5’-GTCACCTTGTGCTGTTTGGCTCCATGTTC-3’. PCR products were cloned into the pEF6V5HisTopo expression plasmid (Invitrogen) with their original start and stop codons intact. For generating a cDNA clone encoding WUCAM fused with the FLAG epitope tag at the Nterminus (WUCAM^FLAG), an additional primer (5’-ACTTAGAAGCTTGCCTCAGGAATGATGACAGGCAC-3’) was used to introduce a Hind III restriction enzyme site after the leader sequence to allow sub-cloning into the pFLAGCMV3 vector (Sigma) in addition to an Xba I site picked up from pEF6V5HisTopo. Similarly for Tage4^FLAG, a primer (5’-TAGTAGGCGGCCGCGGGTGGGGAGATAGCTGTGCAG-3’) was used to introduce Not I restriction enzyme site after the leader sequence to allow sub-cloning into the pFLAGCMV3 vector (Sigma) in addition to a Not I site at the 3’ end picked up from pEF6V5HisTopo. A chimeric soluble molecule comprising the Ig domain of WUCAM and the Fc region of mIgG1 was produced by overlap/extension PCR with the following primers 5’-CACGGTGCCAGGGATTGTGGTTGTAAGCCTTGC-3’ and 5’-CAACCACAATCCCTGGCACCGTGCTCAGCCAC-3’. Baf3 cells were transfected with 13 WUCAM^FLAG cDNA or pFLAGCMV3 vector alone by electroporation and cultured in selective medium containing neomycin. Cell clones expressing WUCAM^FLAG were selected by flow cytometry with the M2 anti-FLAG antibody (KODAK) and sorted for high expression. PVR and other Nectins/Necls were previously cloned and expressed in Daudi, P815 or Baf3 [14, 17]. Human Fc (IgG1) or mouse Fc (IgG1) fusion proteins were made as reported previously [14, 17].

Antibodies

MAbs 4E1.2 (mouse IgG3) was generated by immunizing BALB/c mice with WUCAM^FLAG-Baf3 cells, as previously described [14]. Anti-PVR (clone SKII.4), mouse IgG1, was previously generated [14]. An irrelevant IgG3 control antibody was obtained from ATCC (CCL-189, anti-influenza virus). Biotinylated anti-mouse IgG3 antibody was from Pharmingen BD. FITC and PE-labeled antibodies to CD3, CD4, CD8, CD56, CD19, CD25, CD45RA, CD45RO and CD69 were from Beckman Coulter. CXCR5 (mouse IgG2b) was obtained from R&D. Anti-human/mouse ICOS was purchased from eBioscience.

Cell conjugation, binding and laminar flow assays

Conjugate and binding assays were performed essentially as described [17]. For conjugates assays between different mouse cell lines, cells were either labeled with CFSE, DiO, DiL or DiD (Vybrant Multicolor Cell-Labeling Kit, Invitrogen). Human cells were identified in conjugates by staining with anti-human CD45-APC (Beckman Coulter). Laminar flow assays were performed exactly as previously described [17].

Cell Cultures

PBMC were prepared from buffy coats, according to described procedures. Tonsils were obtained from Children’s Hospital, Washington University School of Medicine, St. Louis, M. Approval was obtained from the Washington University School of Medicine institutional review board for these studies. CD4⁺ T cells were pre-enriched from tonsils using CD4 magnetic beads (Miltenyi Biotec).

Immunohistochemistry

Specimens from human tissues included reactive lymph nodes and spleen removed for diagnostic purposes. Cryostat sections of frozen specimens were air dried overnight at room temperature and fixed in acetone for 10 minutes before staining. PVR was detected with mAb SKII.4, followed by biotinylated anti-immunoglobulin multilinks secondary antibody (Biogenex, San Ramon, CA) and streptavidin-immunoperoxidase or streptavidinealkaline-phosphatase.