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First published online 7 August 2007
doi: 10.1242/jcs.006361

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Tetraspanin CD81 is required for the v5-integrin-dependent particle-binding step of RPE phagocytosis

¹ Dyson Vision Research Institute, Department of Ophthalmology, Graduate Program in Physiology, Biophysics and Systems Biology, Weill Cornell Medical College, New York, NY 10021, USA
² Dyson Vision Research Institute, Department of Ophthalmology, Department of Physiology and Biophysics, and Department of Cell and Developmental Biology, Weill Cornell Medical College, New York, NY 10021, USA

View larger version (76K): [in this window] [in a new window]   Fig. 1. CD81 and CD9 are abundantly expressed at the apical, phagocytic surface of RPE cells. (A,B) Polarized live RPE-J cells on ice were labeled with CD81 or CD9 antibodies as indicated and shown in green, subsequently fixed and labeled with ZO-1 tight junction protein antibody (red). Nuclei are shown in blue in the merged fields. Maximal projections of representative whole cell x-y scans acquired at 0.2 µm intervals are shown. (C-H) Representative confocal x-z scans of polarized RPE-J cells co-labeled with antibodies to CD81, CD9, ZO-1, 1 integrin, P-cadherin or v5 receptors as indicated. CD81, CD9 and v5 were labeled in live cells on ice as in (A,B). ZO-1, 1 integrin and P-cadherin were labeled after fixation and permeabilization. Single and merged channels of the same field are shown with the labeled proteins indicated above each single channel picture. Bars, 10 µm.

View larger version (132K): [in this window] [in a new window]   Fig. 2. CD81 partially colocalizes with v5 integrin receptors at the apical surface of RPE cells. Live RPE-J cells were labeled with either CD81 antibody Eat2 (left column) or v5 receptor antibody P1F6 (middle column), or both antibodies (right column). All samples received both secondary antibodies and were examined at both corresponding wavelengths to ensure labeling specificity of secondary antibodies. CD81 signals are shown in green in the middle row, v5 signals are shown in red in the top row. The bottom row shows the merged signals of upper and middle rows. Panels show representative single x-y plane confocal microscopy scans of the RPE-J apical surface. Areas with focus on the apical surface of cells showed considerable colocalization of CD81 and v5, appearing in yellow in the merged images. Selected areas are enlarged and shown in the fourth column to better illustrate co-localization. Bars, 10 µm.

View larger version (40K): [in this window] [in a new window]   Fig. 3. CD81 resides in a complex with v5 but not with MerTK or CD36 in RPE cells. (A) Polarized RPE-J cells were separated into extracted (Ex) and insoluble pellet (P) fractions by differential extraction with buffer containing 1% Triton X-100 (Tx), Brij97 (B97), or CHAPS (Ch) either still on ice or with vortexing as indicated. Equal volumes of fractions were analyzed by western blotting with antibodies as indicated to the right of the panels. (B) IPs from vortexed RPE-J Brij97 lysates (as in A) with antibodies to CD81 or phagocytic receptors 5 integrin, MerTK and CD36, and non-immune hamster and goat antibodies were probed sequentially in the order of panels shown (top to bottom) for precipitated receptors and for CD9 as indicated to the left of each panel. CD81 and 5 antibodies co-isolated CD81, CD9, v, and 5. Because after sequential re-probing of blots ezrin antibody showed only very weak ezrin signals, receptor IPs were repeated and fresh blots probed with ezrin yielding robust bands representing co-precipitated ezrin specifically with CD81 and 5 integrin (panel ezrin). (C) Tissue samples of neural retina (NR) and corresponding RPE-choroid (RPE/Ch) isolated from individual 5+/+ and 5–/– mouse eyes were lysed in buffer containing Brij97. IPs from these tissue lysates with CD81 antibody co-precipitated v integrin from 5+/+ neural retina and RPE/choroid and from 5–/– neural retina but not from 5–/– RPE/choroid (RPE/Ch) (v panel). CD81 immunoblotting revealed that CD81 antibody precipitated similar amounts of CD81 from 5+/+ and 5–/– tissues (CD81 panel). Non-immune antibodies did not isolate any of the proteins of interest (data not shown).

View larger version (36K): [in this window] [in a new window]   Fig. 4. Immuno-blocking CD81 mainly decreases surface-binding of POS by RPE cells. (A-D) Rat RPE-J cells were challenged with POS for 3.5 or 5 hours as indicated, in the presence of rat CD9 mAb RPM.7 (gray bars), rat and mouse CD81 mAb 2F7 (black bars), or non-immune IgG (white bars). (E) Human D407 RPE cells were challenged with POS for 5 hours in the presence of human CD81 mAb clone I.3.3.22 (black bars). (F) D407 cells were challenged with POS for 3.5 hours in the presence of human CD9 mAb MM2/57 (gray bar), human CD81 mAb clone I.3.3.22 or JS-81 (black bars a, b, respectively) or non-immune IgG (white bar). Antibody concentrations are indicated in µg/ml below each bar. Pre-incubation with antibody before addition of POS had no additional effect on POS binding (data not shown). Results are presented as mean ± s.d., n=3, of bound (A,C,E,F) and internalized (B,D) POS per RPE cell. In A and B Student's t-test was used to compare samples receiving tetraspanin antibody with samples receiving appropriate non-immune control antibody at the same concentration. Asterisks denote significant differences with P<0.05. (A,B) In 5-hour assays, CD81 antibody significantly inhibited both POS binding (P<0.01) and internalization (P<0.05) by RPE-J cells. CD9 antibody had no significant effect (P>0.05). (C,D) In 3.5-hour assays, CD81 antibody significantly inhibited only POS binding by RPE-J cells (P<0.01). CD9 antibody had no effect (P>0.05) and internalization was unaffected by either antibody (P>0.05). (E,F) Two different CD81 antibodies significantly inhibited POS binding by D407 cells (P<0.02). All other conditions did not cause significant changes (P>0.05). RPE-J cells without antibody bound 2.5 POS and internalized 1.1 POS per cell on average during a 5 hour assay. D407 cells without antibody bound 2.4 POS and internalized 1.4 POS per cell during a 5-hour assay. Hence, non-immune IgG did not significantly alter control uptake.

View larger version (75K): [in this window] [in a new window]   Fig. 5. CD81 depletion diminishes POS binding. RPE-J cells were transfected with either control siRNA (si-control), CD9-specific or CD81-specific siRNA (si-CD9 and si-CD81 respectively). 48 hours after transfection, protein expression (A-D) or phagocytic activity (E,F) of transfectants were evaluated. (A,B) Whole cell lysate representing equal numbers of cells were analyzed by western blotting with antibodies as indicated. (C) Quantification of western blots shows that CD81 and CD9 protein levels were reduced to 0.48±0.11-fold and 0.33±0.02-fold, respectively, by si-CD81 or si-CD9 transfection compared to si-control (mean ± s.d., n=3). (D) Transfectants, as indicated, were fixed with 4% paraformaldehyde before labeling with CD81 or CD9 antibodies (green). X-y maximal projections of representative images were acquired as described for Fig. 1. (E) Transfected cells were challenged with FITC-POS for 3.5 hours. X-y maximal projections of representative images show total FITC-labeled POS (green) taken up by the cells. Cell nuclei are shown in red in D and E. (F) Live transfectants were labeled on ice with v5 antibody P1F6 and fixed before labeling with ZO-1 antibodies. X-z scans of representative areas show ZO-1 only or overlay of v5 and ZO-1 as indicated. Bars, 10 µm. (G) POS phagocytosis assays were analyzed by fluorescence scanning to quantify bound and internalized POS. Bars represent mean ± s.d., n=3, bound and internalized POS per RPE cell as indicated (white bars: si-control; gray bars: si-CD9; black bars: si-CD81). CD81 siRNA significantly reduced the numbers of bound POS compared to si-control (asterisk, Student's t-test, P<0.02). CD9 siRNA had no significant effect (P>0.05).

View larger version (43K): [in this window] [in a new window]   Fig. 6. CD81 overexpression increases POS binding by RPE cells. RPE-J cells were transfected with plasmids encoding mouse CD81 (mCD81), CD9 (mCD9), or empty vector as indicated. 48 hours after transfection, tetraspanin expression (A-D) and phagocytic activity of transfectants (E) were evaluated. (A) RPE proteins, as indicated, were analyzed by immunoblotting of transfectant lysates from equal numbers of cells. CD81 antibody Eat2 recognized both endogenous rat and transfected mouse CD81 protein. Mouse CD9 antibody KMC (mCD9) and rat CD9 antibody RPM.7 (rCD9) recognized exogenous mouse and endogenous rat CD9, respectively. (B) Quantification of western blots shows that CD81 transfection increased CD81 protein level to 2.1±0.4 fold of empty vector transfected cells (mean ± s.d., n=3). mCD9 plasmid transfection did not change CD81 expression. (C) Transfected CD81 and CD9 localized to the apical surface of transfected RPE-J cells. Cells were fixed with 4% paraformaldehyde before labeling CD81 and mouse CD9 (mCD9) as indicated (green). Panels show x-y maximal projections acquired as described for Fig. 1 of representative fields. (D) Transfected cells were challenged with FITC-POS for 3.5 hours. Maximal projections of representative fields show total (bound plus internal) FITC-POS (green) taken up by the cells. Cell nuclei were stained with DAPI (red) in C and D. Bars, 10 µm. (E) 3.5-hour POS phagocytosis assays were analyzed by fluorescence scanning to quantify bound and internal POS. Bars represent mean ± s.d., n=3, bound POS and internalized POS per RPE cell transfected with empty vector (white bars), CD9 (gray bars), or CD81 (black bars) expression plasmids. CD81 overexpression significantly increased the numbers of bound POS but not of internal POS as compared to vector control (asterisk, Student's t-test, P<0.03). CD9 transfection had no effect (P>0.05).

View larger version (53K): [in this window] [in a new window]   Fig. 7. CD81 but not CD9 overexpression specifically increases surface levels of v5 integrin receptors. Cells transfected with empty plasmid (p), CD81 or CD9 expression plasmids as indicated, (tf:) were either incubated live on ice with antibodies to receptor proteins for live surface IPs (left 3 lanes) or lysed and subjected to total lysate IP (right 3 lanes). Live surface and total lysate IPs were analyzed on the same gel and immunoblotted. Antibodies used for IP and immunoblotting detection are indicated to the left of each panel (IP>blot:). The experiment was performed four times independently. Band intensities of tetraspanin-overexpressing cells were compared with band intensities obtained from empty plasmid controls for each experiment and results averaged. CD81 transfection significantly (P<0.05) increased surface CD81 by 58±13%, total CD81 by 83±11%, surface v by 36±5%, and surface 5 by 31±9% (mean ± s.d., all n=4). All other bands did not differ significantly from control IPs (average change <10%, P>0.05). Non-specific antibodies did not isolate any of the proteins tested in surface or lysate IPs (data not shown).

View larger version (51K): [in this window] [in a new window]   Fig. 8. CD81 antibody inhibits POS binding by 5+/+ mouse RPE and RCS (MerTK-deficient) rat RPE but not by 5–/– mouse RPE, whereas CD9 antibody has no effect. (A-F) Live, polarized, primary RPE from 5+/+ mice, 5–/– mice, and RCS rats were labeled with CD81 (A-C) or CD9 (D-F) antibody, fixed, and labeled with ZO-1 antibody to visualize tight junctions. A-F show x-y maximal projections acquired by confocal microscopy as described for Fig. 1. CD81 and CD9 apical surface labeling did not differ between samples prepared from animals of different genotype. (G,H) Total FITC-POS uptake by 5+/+ primary RPE after 1 hour of POS challenge was visibly reduced by CD81 antibody but not by non-immune (n.i.) antibody at 50 µg/ml. Representative fields show maximal x-y projections of FITC-POS, nuclei, and ZO-1 tight junctions. Bars, 10 µm. (I) Primary RPE cells were challenged with FITC-POS for 1 hour in the presence of non-immune (n.i.) IgG (white bars), CD81 (black bars) or CD9 (gray bars) mAbs at 20 or 50 µg/ml as indicated below each bar. Antibodies used were CD81 mAb clone Eat2 and 2F7, anti-mouse CD9 mAb KMC, anti-rat CD9 mAb RPM.7. Fluorescence scanning quantification of bound FITC-POS shows that CD81 antibodies inhibited POS binding by 5+/+ RPE and RCS rat RPE, but had no effect on the residual POS binding by 5–/– RPE (black bars). By contrast, CD9 antibodies did not change POS binding by any of the three types of RPE (gray bars). Bars represent mean number of POS bound per RPE cell ± s.d., n=3. Asterisk denotes significant inhibition of POS binding compared to appropriate non-immune antibody control at the same dose (P<0.01, Student's t-test). None of the conditions affected POS internalization (data not shown).

View larger version (66K): [in this window] [in a new window]   Fig. 9. CD81 overexpression promotes POS binding only in the presence of v5 integrin. Primary RPE isolated from 5+/+ mice, 5–/– mice, and RCS rats were transfected with CD81 expression plasmid or empty vector as control. 48 hours after transfection, we evaluated protein expression (A,B) and phagocytic activity (C) of transfectants were examined. (A) Total cell lysates, of equal numbers of cells, were analyzed by western blotting for proteins as indicated. (B) Quantification of western blots shows that CD81 plasmid transfection specifically increased CD81 protein to 1.4-1.6 fold of control cells. Bars show relative CD81 levels in 5+/+ (black), 5–/– (dark gray), and RCS RPE (light gray) (mean ± s.d., n=3). (C) Quantification of surface-bound POS following 1 hour of FITC-POS challenge shows that CD81 overexpression increased POS binding by 5+/+ and RCS RPE, but had no effect on 5–/– RPE. Bars represent mean ± s.d., n=3, percentage POS bound by CD81 overexpressing RPE cells compared to control transfected cells of the same genotype. POS binding by CD81 transfected 5+/+ and RCS RPE was significantly increased compared to respective control uptake (P<0.05, Student's t-test). Remarkably, CD81 transfected 5–/– RPE did not differ in POS binding from control 5–/– cells (P>0.5).

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