EXPERIMENTAL

CELL

RESEARCH

203,

182-191

(1992)

Generation of Anti-idiotypic Monoclonal Antibodies Recognizing Vasopressin Receptors in Cultured Cells and Kidney Sections MIREK

JURZAK,

Max-Planck-Institut

DAVID A. JAN&*

WINFRIED

HAASE,

Inc.

INTRODUCTION Two peripheral G-protein-coupled receptor subtypes for the neurohypophyseal nonapept,ide hormone vasopressin have been described. The Vi-receptor type, found in mammalian liver and in smooth muscle, activates the phosphatidylinositol-signal pathway and moand vabilizes internal Ca ‘+ , leading to glycogenolysis soconstriction [ 11. The renal V,-receptor mediates anti-

’ To whom correspondence and reprint requests should be addressed at Max Planck Institut fiir Biophysik, Kennedy Allee 70, D-WOO Frankfurt am Main 70, Germany. Fax: 004969/6303244. 0014-4827/92

Copyright All

rights

AND FALK FAHRENHOLZ’

fiir Biophyszk, Kennedy Allee 70, D-6000 Frankfurt am Main 70, Germany; *Institut fiir Medancsche Westftilische Wilhelm+Uniuersit& Robert-Koch-Strasse 31, 4400 Miinster, Germany

To produce anti-idiotypic antibodies against receptors for the neurohypophyseal hormone vasopressin, an anti-vasopressin monoclonal antibody with a ligand specificity similar to that of vasopressin receptors was employed for immunization. Three anti-idiotypic monoclonal antibodies were obtained which induced, like vaplasminogen activator production in the sopressin, renal epithelial cell line LLC-PK, (expressingvz-receptors). Induction of plasminogen activator synthesis by the anti-idiotypic antibodies could be inhibited by coincubation with a vasopressin antagonist. In a fashion similar to that of vasopressin itself, the anti-idiotypic antibodies induced receptor down-regulation. The anti-idiotypic antibodies were employed to visualize vasopressin receptors on LLC-PK, and A7r5 (V,-receptor-expressing) smooth muscle cells by immunofluorescence. Antibody-mediated fluorescence was not observed in receptor-deficient mutant cell lines or vasopressin-receptor-down-regulated cells. Furthermore, these antibodies were used for immunohistochemical localization of vasopressin receptors in rat and bovine kidney preparations. In accordance with earlier physiological and biochemical observations, vasopressin receptors were detected predominantly in collecting ducts in cortex and medulla. On the cellular level, a differential staining pattern was observed. (~3 1992 Academic Press,

REINER PETERS,*

$5.00

0 1992 hy Academic Press, Inc. of reproduction in any form reserved.

Physck,

diuresis by activation of adenylate cyclase in the distal parts of the nephron [2]. The low level of vasopressin receptors and the lack of hormone binding activity after solubilization [3, 41 has prevented efficient purification of vasopressin receptor proteins. Therefore, a major goal, the production of receptor-specific antibodies, could not be achieved by direct immunization with purified or highly enriched receptor proteins. In this study we applied an alternat,ive approach: the anti-idiotypic route to receptor-specific antibodies. This approach is based on Jerne’s network hypothesis [Ei], wherehy anti-ligand antihodies with receptor-like binding properties are used to induce antibodies which cross-react with the ligand’s receptor. Various recept,or systems have been studied since then using this approach (for review see [6, 71). For the induction of monoclonal vasopressin-receptor-specific anti-idiotypic antibodies the monoclonal anti-vasopressin antibody mab’ 113 [8] was used. As shown by epitope analysis, mab 113 preferentially recognizes the C-terminal portion of vasopressin (Pro-ArgGly-NH,) and partly displays the ligand specificity of vasopressin receptors. Both mab 113 and vasopressin receptors bind vasopressin analogues only if the molecule carries a positive charge in position 8 and a GlyNH, moiety in position 9. The substitution of leucine at position 8, found in oxytocin, leads to complete loss of affinity. Mab 113 binds both VI-receptor antagonists and V,-receptor-specific agonists [S]. The LLC-PK, pig kidney epithelial cell line [Y], which possesses V,-receptors, was used for screening. In LLCPK, cells agonistic stimulation of the CAMP system leads to production of the extracellularly secreted protease urokinase-type plasminogen activat,or (uPA). This provided the basis for a simple and novel screening test in solid media for receptor-cross-reacting anti-idiotypes. V,-receptor-deficient cell lines (Ml8 and VPRl)

* Abbreviations used: TR-dLVP, 1-deamino[8-lysine(l-tetramethylrhodamylaminothiocarbonyl)] vasopressin; mab, monoclonal antibody; BSA, bovine serum albumin; uPA, plasminogen activator; FITC, fluorescein isothiocyanate.

182

ANTIBODIES

TO VASOPRESSIN

were available as negative controls [lo, 111. The A7r5 cell line, derived from rat aorta and expressing the smooth muscle V,-receptor [ 121, was used as a model of the Vi-receptor system. By this means it was possible to obtain monoclonal anti-idiotypic antibodies with specificity to vasopressin receptors. Furthermore, the anti-idiotypic antibodies could be employed to visualize vasopressin receptors in both cultured cells and histological sections from kidney, one of the major target organs of vasopressin. EXPERIMENTAL

PROCEDURES

Analogues of uasopressin. The V,/V,-specific vasopressin antagonist d(CH,),[D-Phe’, Ile4,Lys-NH,g]AVP [13] was prepared by solid phase synthesis as described previously [14, 151. 1-Deamino[8-lysine(A)‘-tetramethylrhodamylaminothiocarbonyl)] vasopressin (TRdLVP), a rhodamine-labeled analogue ofvasopressin [ 161, was synthesized as described [ 171. Immunization and cell fusion procedure. The monoclonal idiotypic antibody mah 113 (81 derived from Balh/c mouse was purified from ascites fluid by gel filtration on a Sephacryl S-300 C-26/100 column (Pharmacia, Freiburg). Six-week-old female Balh/c mice were immunized by intraperitoneal injection of 100 gg purified antibody 113 in NaClIP, and 50% complete Freund’s adjuvant (GIBCO, Eggenstein). A booster injection was given ip 4 weeks later in 50% incomplete Freund’s adjuvant. Six weeks after the booster injection and 3 days before cell fusion the best-responding mouse was injected with 150 pg antibody in NaClIP, intraperitoneally. The spleen cells were fused with the X-63-Ag8-653 myeloma cell line (Flow, Meckenheim) following a modified protocol of Lane et al. [18] and Fazekas de StCroth and Scheidegger [19]. Isotypes of the monoclonals were determined using a immunodiffusion isotyping kit (ICN, Eschwege). Ascitic fluid was prepared according to Harlow and Lane [20]. IgM containing control ascites ABPC 22 was obtained from Sigma (Deisenhofen). Cell culture. The myeloma and hybridoma cells were grown as previously described [8]. Cells and mutants of the LLC-PK, pig kidney epithelial cell line [9] and the A7r5 rat smooth muscle cell line [12] were cultured as previously described [17, 211. IP, determinations in A7r5 cell monolayers were performed as described 1211. Assay of antisera in ELISA and in competitive binding tests on rat Blood samples, taken 10 days after booster injecliver membranes. tion, were assayed in an ELISA procedure as described previously [8] except that mab 113 was used to coat the microtiter plates. All assays were performed in duplicate and the absorbance was measured at 450 nm in a Titertek multiscan photometer (Flow). Rat liver membranes were prepared and [3H]vasopressin (NEN, Dreieich; 70 Ci/mmol) binding to the Vi-receptor was measured as described [22] with the following modifications: 100 fig membranes in binding buffer was preincubated with NaCl/P,-diluted mouse anti-idiotypic antisera for 2 h on ice. After the addition of [3H]vasopressin (lo-‘M) the samples were incubated for a further 30 min at 30°C and hound radioactivity was determined. Nonspecific binding was determined in the presence of 1O-6 M nonlabeled vasopressin. Nonimmune serum served as control, with all assays performed in duplicate. Primary screening of hyScreening of hybridoma supernatants. hridoma supernatants was performed using the idiotype-ELISA and membrane binding competition assay described above for antisera. Secondary screening of anti-idiotypic clones was performed using a modified functional uPA assay [23] on LLC-PK, cells. The cells grown in 24.well plates to 90% confluency were treated with hybridoma supernatant or diluted ascites (in DMEM-0.1% BSA) for 223 h at 37°C and washed three times with NaClIP, and the antibody-containing solution was replaced by 300 ~1 DMEM-0.1% BSA. After 5-16

183

RECEPTORS

h of incubation at 37”C, induced “PA-activity was detected by spotting 5 +l of the conditioned medium onto agar of 1.36% casein, 0.85% agarose, and 0.014 mg/ml plasminogen [12]. Lytic zones of proteolysis indicative of uPA production were observed after 2-3 h at 37°C. Nonconditioned media, control ascites (Renner, Dannstadt), and antibody-treated cells of the V,-receptor-deficient cell line Ml8 served as negative controls. Positive clones were selected for furtherpropagation and characterization. Monoclonal antibodies were purified from ascites fluids: mab 237 and 290 which are of IgM type by gel filtration or anion-exchange chromatography and mab 255 (IgG, type) by protein A chromatography. Cells were grown on coverslips (15 X Fluorescence measurements. 15 mm) for 3-4 days to 50-70% confluence. After incubation with the first antibody (ascites diluted 1:50 in DMEM-0.1% BSA, 10 mM Hepes, pH 7.3) for 30 min at 37”C, cells were washed with NaClIP, (containing 0.5 mg/ml BSA) and fixed for 10 min with 4% paraformaldehyde in NaClIP, at RT or with cold acetone for 2 min on ice. Cells were then incubated for 1 h in 1% glycine in NaClIP,, washed, incubated for 1 h at room temperature with FITC-labeled antiserum (Sigma) diluted 1:lOO in NaClIP,-1% BSA, and washed and mounted in NaCl/Pi-1 % BSA. Cells preincubated overnight with 10m6M vasopressin prior to antibody incubation or receptor-deficient mutants served as a negative control. Quantitation of the fluorescence intensity was performed using a microphotolysis apparatus descrihedpreviously [24]. For the study of antibody-induced receptor down-regulation, cells were preincuhated overnight with diluted ascites (1:50) of anti-idiotypic antibodies or hormone (1O-7 M). After washing, the incubation with TR-dLVP was performed for 30 min at RT, the cells were washed again and mounted, and the cell-associated fluorescence was measured as described [17, 211. Immunohistochemistry. Rat (Wistar) kidneys or bovine papilla were fixed by immersion in Bouin fixative (9.5% formaldehyde in picrinic acid with 6.25% acetic acid) for 2-24 h, dehydrated in ethanol, and embedded in paraffin. Dewaxed sections (5 pm) were rehydrated and incubated in 0.5 M ammonium chloride or 2% glycine in phosphate buffer (pH 7.3). The anti-idiotypic antibodies were used as ascites fluid in NaClIP, diluted in 1% BSA, 0.2% Tween 20, and 0.1% Triton X-100 after 1 h preincubation with buffer alone. Antihody binding sites were visualized by peroxidase-coupled secondary antiserum (rabbit anti-mouse-POD, Sigma) with diaminobenzidine or 3amino-9-ethylcarbazole as peroxidase substrates [25]. In control experiments first antibodies were omitted.

RESULTS

Screening

of Antisera

for Anti-idiotypic

Reaction

Five mice were immunized syngeneically with purified monoclonal idiotypic IgA antibody mab 113 and sera screened both for anti-idiotypic reaction with mab 113 using an ELISA and for inhibition of vasopressin binding on Vi-receptor-rich rat liver plasma membranes. All sera exhibited a high titer to mab 113 compared to that of a nonimmune serum. In the serum with the highest antibody titer half-maximal reaction was observed at 400,000-fold dilution (not shown). The inhibition of specific [3H]vasopressin binding caused by this serum was concentration dependent and amounted to 50 and 30% at a dilution of lOO- and lOOO-fold, respectively (Fig. 1). The binding of [3H]prazosin, an 01~adrenergic antagonist, was negligibly influenced (not shown). These results indicated the presence of anti-

184

JURZAK

-

I 1

-

J

FIG. 1. Inhibition of [3H]vasopressin binding to rat liver V,-vasopressin receptor by an anti-idiotypic mouse serum. Rat liver plasma membranes (100 fig) were preincubated with the NaCl/P,-diluted mouse anti-idiotypic antiserum showing the highest titer to idiotypic antibody mab 113. After addition of [“Hlvasopressin (10-s M) the samples were incubated for a further 30 min at 30°C and bound radioactivity was determined. Nonspecific binding was determined in the presence of 10m6M nonlabeled vasopressin. Nonimmune serum served as control.

ET AL.

gen-containing agar [lo], where the proteolytic activity induces lytic zones [23]. Of the supernatants tested, nine were capable of stimulating uPA production in LLC-PK, cells. Antibody-induced CAMP production could not be measured, indicating the superior sensitivity of the uPA assay for detecting the partial agonistic properties of these antibodies. As an example, Fig. 2 shows uPA induction by mab 237, mab 234, and mab 101, in contrast to the control myeloma supernatant (Myeloma) and the negative clone 154. Like the hormone itself, none of these antibodies triggered uPA production in the V,-receptordeficient LLC-PK, mutant Ml8 cell line (indicated by I18 in Fig. 2 and not shown). The receptor-independent adenylate cyclase activator forskolin induced uPA production in both cell lines, indicating an intact signal transducing cascade distal to the receptor in the mutant (Fig. 2) consistent with previous observations [lo]. Further evidence for V,-receptor specificity of the agonistic effect of the anti-idiotypic antibodies was obtained by incubation of the cells with antibodies together with

idiotypic antibodies with specificity for the vasopressin receptor in the investigated serum.

Screening

for Monoclonal

Anti-idiotypic

Antibodies

Supernatants of 475 clones of 1008 seeded wells were screened for reactivity to plate-bound idiotypic antibody by ELISA as described for the antisera. One hundred thirty-nine supernatants were positive, giving rise to more than double the background reaction displayed by the supernatant of the parental myeloma cell line. Binding inhibition assays on the V,-receptor-containing bovine kidney and on Vi-receptor-containing rat liver membranes proved ineffective as a screening system to detect anti-vasopressin receptor anti-idiotypes because of the extreme instability of the receptors under the incubation conditions, resulting in a loss of maximal [3H]vasopressin binding even in the presence of unconditioned medium. Accordingly, a novel functional assay was used, exploiting the LLC-PK, renal epithelial cell line, which possesses V,-receptors coupled to adenylate cyclase stimulation. Vasopressin-mediated elevation or other agonist-mediated elevation of intracellular CAMP levels in LLC-PK, cells leads to the production of uPA. The extracellularly secreted protease can be detected by spotting conditioned media onto a casein and plasmino-

FIG. 2. Plasminogen activator (“PA) production induced by monoclonal anti-idiotypic antibodies on LLC-PK, cells. Cells were treated with hybridoma supernatants, washed, and incubated in DMEM-0.1% BSA. Induced proteolytic activity was detected by spotting 5 ~1 conditioned medium onto agar plates containing casein and plasminogen. Lytic zones indicate uPA production. 101, 234, and 237 are antibody-producing hybridoma, whereas 154 is a negative clone. Hybridoma supernatant (Myeloma) and the V,-receptor-deficient mutant Ml8 (118) served as negative controls. ANT indicates coincubation with lOma M of the V,/V,-receptor-specific antagonist d(CH,),[D-Phe2,11e4,Lys-NHz9]AVP. The adenylate cyclase activator forskolin and vasopressin (AVP) were used as positive controls.

ANTIBODIES

TO VASOPRESSIN

RECEPTORS

185

the V,/V,-specific antagonist d(CH,),[D-Phe2,11e4,LysNH,‘]AVP (+ANT in Fig. 2). At concentrations as low as 10e8 M the antagonist inhibited uPA production by LLC-PK, cells induced by either lo-” M vasopressin or several of the monoclonal anti-idiotypic antibodies, including mab 237 (Fig. 2), mab 255, and mab 290 (not shown). These three monoclonals were selected for further study, of which mab 237 and mab 290 were determined to be IgMs and mab 255 IgG,. In A7r5 cells which express V, receptors coupled to phospholipase C, antibody-induced inositol 1,4,5-triphosphate (IP,) production was not detectable. Binding

Characteristics

of the Anti-idiotypes

Competition between vasopressin and anti-idiotypic antibodies for binding to the idiotypic antibody mab 113 was measured in a modified RIA. The assay was based on the retention of the idiotypic IgA antibody-[3H]vasopressin complexes on polyethyleneimine-treated glass fiber filters [B]. Compared to control ascites fluid, ascites fluid containing the anti-idiotypic antibodies mab 237,255, and 290 reduced binding of vasopressin by approximately 30%, indicating an interaction of the anti-idiotypic antibodies with the paratope of the idiotype. The three anti-idiotypic monoclonals also inhibited [3H]vasopressin binding to the VI-receptor of rat liver plasma membranes compared to control ascites. At a lo-fold dilution mab 237 and mab 255 ascites inhibited hormone binding by 45 and 30%, respectively. Purified antibodies mab 237, 255, and 290 inhibited at 0.67 pg antibody per milliliter [3H]vasopressin binding by 15 to 28%. The molar concentration of purified antibodies was 6.7 X lOpa M for IgM (237 and 290) and 4.5 X lop7 M for IgG. IgM-containing control ascites had no effect on [3H]vasopressin binding. Competitive inhibition assays of [“Hlvasopressin binding to the V,-receptor in bovine kidney membranes were ineffective because of the extreme instability of the V,-receptor under the incubation conditions. Visualization Complexes

of Anti-idiotypic AntibodylReceptor by Confocal Laser Scanning Microscopy

Cells of the LLC-PK, cell line were incubated with anti-idiotypic antibodies for 30 min at 37°C. Antibody binding was then visualized in a confocal laser scanning microscope using a fluorescein-isothiocyanate (FITC)labeled second antiserum. Typical results for mab 290, which are also representative for mab 237 and mab 255 (not shown) are displayed in Fig. 3. Antibody-mediated fluorescence occurred prevalently in the form of small aggregates (Fig. 3A), indicating receptor clustering. Cells preincubated with 10d7 M vasopressin and hence down-regulated for the V,-receptor showed reduced antibody-mediated fluorescence (Fig. 3B). Immunofluorescence was virtually absent from cells of the V,-receptor-

FIG. 3. Visualization of antibody-mediated fluorescence on LLC-PK, cells. Cells were incubated for 30 min at 37°C with mab 290 (diluted ascites fluid), washed, fixed, and incubated with a FITC-coupled second antiserum from rabbit (A). Immunofluorescence was virtually absent from receptor down-regulated LLC-PK, cells pretreated with 10m7Mvasopressin (B) and from cells of the V,-receptordeficient cell line VPRl (C).

deficient cell line VPRl [ll] (Fig. 3C). In experiments performed with LLC-PK, cells at 4”C, a more homogeneous surface distribution of antibody receptor complexes was observed. Similar results were obtained with A7r5 cells which

JURZAK

LLC-PKt

231

was evident for all three anti-idiotypic antibodies for both LLC-PK, and A7r5 cells. Down-regulation of receptor numbers by preincubation of cells with 10m6M vasopressin reduced anti-idiotype-mediated fluorescence to 540% that of the control. The V,-receptor-deficient cell line Ml8 exhibited negligible specific fluorescence (not shown). The microfluorometric methods outlined above were also employed to study the time dependence of antiidiotype binding. It was found (Fig. 5) that mab 290-induced specific fluorescence on LLC-PK, cells reached a maximum after 30 min of the incubation with the antiidiotype. At longer incubation times fluorescence decreased, arriving at zero at about 120 min. This suggests receptor-mediated internalization of surface-bound anti-idiotypic antibody.

A7r5

h

I 255

1 I L 290 monoclonal

237

ET AL

255

290

antibody

FIG. 4. Quantification of immunofluorescence of anti-idiotype binding to LLC-PK, and A7r5 cells by microtluorometry. Cells were incubated with anti-idiotypes for 30 min at 37”C, washed, fixed, and incubated with a FITC-labeled second antiserum. Fluorescence of the basal plasmamembrane was measured as described under Experimental Procedures. Open bars represent measurements of specific antiidiotype-mediated fluorescence. Filled bars represent measurements on cells down-regulated by overnight preincubation with 10~“Mvas.o pressin. Results represent the average of at least 11 separate measurements, whereby the cell-associated fluorescence intensity for a control ascites antibody was subtracted from that for the respective antiidiotypic antibodies treated in identical fashion. The SEM for the raw data was less than 9.4% for LLC-PK, (n > 11) and 7.6% for A7r5 cells (n > 12) of the value of the mean.

Anti-idiotypic regulation

Antibody Induced Receptor Downon LLC-PK, Cells

Further evidence for antibody-induced receptordown-regulation was obtained by long-term preincubation of the LLC-PK, cells with antibodies prior to measurement of specific binding of TR-dLVP [16], a fluorescent V,-agonist with high affinity (KD = 6.9 X lo-’ M) [ 171 for the V,-receptor on LLC-PK, cells. In these ex-

mab

290

express Vi-receptors at densities comparable to those of the V,-receptor on LLC-PK, cells (not shown). As with LLC-PK, cells antibody-mediated cell-bound fluorescence was greatly reduced in the case of “down-regulated” A7r5 cells. The results indicated that the antiidiotypic antibodies bind specifically to LLC-PK, and A7r5 cells, implying recognition of both vasopressin receptor subtypes. Quantification of Anti-idiotypic Microfluorometry

Antibody

Binding

by

The relative amounts of anti-idiotypes binding to vasopressin receptors in the plasma membrane of LLCPK, and A7r5 cells were determined by laser microfluorometry. Cells were incubated with anti-idiotypes, washed, fixed, and then incubated with a FITC-labeled second antiserum as detailed under Experimental Procedures. A microfluorometer [27] was employed to measure fluorescence in small circular areas (2-pm radius) of the basal portion of the plasma membrane. Specific fluorescence was derived by subtracting the fluorescence of cells treated with control ascites fluid from that of cells treated with anti-idiotype-containing ascites fluid. Figure 4 shows typical results. Specific binding

0

30

Incubation

60

120

trme (min)

FIG. 5. Time dependence of antibody binding to LLC-PK, cells. Cells were washed and fixed after different times of incubation with mab 290 at 37°C. Membrane-associated total fluorescence was measured by microfluorometry after labeling with a FITC-coupled second antiserum. Nonspecific fluorescence, determined with vasopressinpreincubated cells, was subtracted from total fluorescence to yield specific fluorescence. Each value represents the average of at least 10 separate measurements, with the SEM indicated being not greater than 8% of the mean value.

ANTIBODIES

TO VASOPRESSIN

FIG. 6. Binding of a rhodamine-labeled vasopressin analogue (TR-dI,VP) to LLC-PK, cells after down-regulation with anti-idiotypic antibodies. Cells were incubated overnight at 37°C with antibodies or 10e7 M vasopressin. After washing, cells were treated with TRdLVP (3 x lo-@ M) for 30 min at 37°C in serum-free medium and fluorescence was measured as described in the legend to Fig. 4. Results represent the mean of more than 10 separate determinations, with the SEM indicated.

periments the cell-associated fluorescence due to TRdLVP binding was quantitated (Fig. 6), whereby preincubation of cells with the anti-idiotypic antibodies led to a substantial decrease of specific TR-dLVP labeling consistent with V,-receptor down-regulation. The idiotypic antibody mab 113, used as a negative control, had no down-regulation effect. Comparable results were obtained by measuring specific [3H]vasopressin binding to LLC-PK, cells after pretreatment with anti-idiotypic antibodies (not shown). Immunohistochemical Labeling Receptors in Rat and Bovine

of Vasopressin Kidney

The monoclonal anti-idiotypic antibodies were employed in light microscopic immunohistochemistry on rat and bovine kidneys. Preferential binding to collecting tubules was observed on tissue sections which had been fixed in Bouin’s fluid whereby typical results for mab 237 and 255 are shown in Fig. 7. In the cortex and the outer medulla of the rat kidney (a-c), where collecting tubules contain principal and intercalated cells, immunolabeling resulted in a differential reaction. In some cells the whole cytoplasm except the nucleus was labeled, whereas other cells showed a prominent peripheral staining of the plasmalemma (Fig. 7~). Cells were

187

RECEPTORS

also observed where antibody binding was restricted to the luminal cell side (Figs. 7b and 7c, solid arrows). In addition, a few cells of collecting tubules in cortex and outer medulla were not stained (Fig. 7c and inset, open arrows). In rat papilla (Fig. 7d) and bovine papilla (Figs. 7e7h), tubules consist of a morphologically homogeneous cell population and were stained by both antibodies. In both species, antibodies also bound to the surface epithelium (E) of the papilla (Figs. 7d, 7e, and 7g; E), which is continuous with the collecting tubule epithelium at the papillary tip. Both antibodies bound predominantly to collecting ducts with no labeling of proximal tubules or of the thick ascending limbs of Henle’s loop. Some reactivity however was observed in rat glomerulae (Figs. 7a and 7b; arrowheads), which may be ascribed to mesangial or capillary cells. Outside the glomerulum, a prominent arteriole is apparently heavily labeled (Fig. 7a; double arrow). The intensely labeled area at the vascular pole (Fig. 7a) indicates either binding of the antibody to elements of the juxtaglomerular apparatus or binding to tangentially sectioned cells of the arteriole. Weak staining of blood vessels in other areas of rat kidney was detected (Fig. 7; crossed arrow). In bovine papilla thin capillaries (V) showed antibody binding (Figs. 7f and 7g). Mab 290 showed similar results (not shown). Antiidiotypic antibody binding to fixed rat and bovine kidney sections was not effectively suppressed by preincubation with vasopressin. This observation could be explained by a modification of the hormone binding site due to the fixation procedure and indicates differences in the binding requirement for hormone and anti-idiotypic antibodies.

DISCUSSION

Activation of the CAMP-dependent hormone-stimulated signal pathway in LLC-PK, cells was employed as a novel screening method, receptor activation being one of the criteria for identifying anti-idiotypic antibodies. Due to the amplification of the CAMP-dependent hormonal signal, the uPA assay allows us to demonstrate the activity of partial V, agonists. V,-vasopressin receptor specificity of these antibodies was shown by lack of the response with receptor-deficient mutants and by coincubation with a V,/V,-specific antagonist. The agonistic function of anti-idiotypic antibodies has been described in other receptor systems, such as the receptors for insulin [27], for P-adrenergic ligands [28], for TSH [29], and for substance P [30]. On A7r5 cells, the antiidiotypic antibodies did not activate the Vi-receptormediated phosphoinositide pathway. The anti-idiotypic antibodies characterized in greater

JURZAK

ET AL.

ANTIBODIES

TO VASOPRESSIN

detail (mab 237, mab 255, mab 290) were able to effect a concentration-dependent inhibition ofvasopressin binding, both to the idiotypic antibody and to the Vi-receptor in rat liver membranes, with an extent of inhibition comparable to that described for other anti-idiotypic antibodies [31, 321. Bjercke and Langone [32] reported that the inhibiting effect caused by anti-nicotine antiidiotypic antibodies (20% at, 0.67 mg/ml) could be enhanced using F(ab’), fragments of the antibodies (50% at comparable concentration). Steric and kinetic factors may restrict a greater extent of binding inhibition of the high-affinity ligand vasopressin by antibodies particularly of the IgM class. The anti-idiotypic antibodies described in this study are directed against epitopes in or close to the vasopressin binding site, which has been shown to be very detergent sensitive [3,4]. Even solubilization with nonionic mild detergents like digitonin induces a loss of hormone-binding properties of vasopressin receptors (unpublished observation). This detergent sensitivity will make immunoprecipitation of solubilized vasopressin receptors with mabs directed against the hormone binding site more difficult. In some Western blot experiments with membranes from bovine kidney and rat liver anti-idiotypic antibody reaction was obtained in a molecular weight range of 30 to 35 kDa, which is consistent with results from photoaffinity labeling experiments [14, 221. However, it proved to be complicated to find reproducible conditions, suggesting that reconstitution of the three-dimensional structure of the epitope after blotting is necessary. The binding of the anti-idiotypic antibodies to vasopressin receptors on intact cells could be visualized using confocal microscopy where specificity for the Vi-receptor of A7r5 cells and the V,-receptor on LLC-PK, cells was shown. Negligible reaction was observed with cells down-regulated by pretreatment with vasopressin or with V,-receptor-deficient LLC-PK, cell lines (Ml8 and VPRl). The specificity for Vi- and V,-receptors was further demonstrated by quantification of fluorescence resulting from antibody binding followed by binding of FITC-labeled second antiserum. Exposure of both cell lines to vasopressin results in receptor-mediated ligand internalization and desensitization [33,34]. The disappearance of surface-bound antibodies from the basal plasma membrane of LLC-PK, cells with time at 37°C was consistent with the time course of hormonal internalization by living LLC-PK,

189

RECEPTORS

cells [ 171. It has been shown that 60 min after binding of [3H]vasopressin at 37°C only 36% of cell-associated ligand remains surface bound. Incubation of LLC-PK, cells with mab 290 for 60 min under the same conditions resulted in 29% fluorescence of that bound after 30 min. The extent of receptor-mediated internalization induced by vasopressin and anti-idiotypic antibody appears to be quantitatively comparable. The finding that long-term incubation of the cells with the anti-idiotypic monoclonals led to a decrease of TR-dLVP binding sites indicates that the antibodies induce receptor internalization and subsequent, down-regulation. That this loss of binding is due to V,-receptor down-regulation and not simple masking of the binding sites by the antibodies can be deduced from the fact that surface-bound antibody is no longer detectable after 2 h of incubation. Results for [3H]vasopressin binding after preincubation of the cells with antibody and a subsequent washing and incubation period in the absence of antibody support this conclusion. Induction of down-regulation by receptor-specific antibodies has been previously described in the transferrin-receptor system [ 351. Specific binding and receptor-mediated internalization of the anti-idiotypic monoclonal antibodies by cell lines expressing both receptor subtypes and their V,-agonistic action on LLC-PK, cells is consistent with “internal image” properties expected for anti-idiotypes of the Ab2p-subtype ]361. Until now immunolocalization of vasopressin receptors with antibodies prepared by direct immunization with the receptor protein has not been possible. The immunochemical staining of rat kidney sections obtained with our anti-idiotypic monoclonal antibodies is in several aspects comparable to that reported by Ravid et al. [37] and Fadool and Aggarwal [38] with predominant labeling of collecting duct epithelial cells. These authors incubated tissue with vasopressin followed by anti-vasopressin antibodies to detect vasopressin-occupied receptors. However, we did not detect labeling of distal convoluted tubules, which was observed by others only after incubation with high doses of vasopressin [37] or in kidney sections from water-deprived rats [38]. In functional tests Woodhall and Tisher [39] found no evidence for a vasopressin response in the distal convoluted tubule. The immunolabeling results indicate that almost all collecting tubule cells are stained although in different subcellular compartments. Our results support the phys-

FIG. 7. Light microscopic immunostaining of rat (a-d) and bovine (e-h) kidneys with anti-idiotypic Antibodies bind to glomerular arterioles (a) and capillaries (a, b; arrowheads) and to collecting ducts (C) in inset) and papilla (d-h). Small vessels are also reactive (c; crossed arrow). Collecting duct cells with membrane are indicated by solid arrows (b, c), unreactive cells by open arrows (c). The surface epithelium (V) are labeled by both antibodies. (a, b) Rat kidney cortex, (c) rat kidney outer medulla, (d) rat papilla,

antibodies 237 (a-f) the cortex (b), outer labeling restricted (e-h) of the papilla (e, h) bovine papilla.

and 255 (g-h). medulla (c and to the luminal and capillaries

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JURZAK

iological observations [40-421 that both cell types of cortical collecting tubules, principal and intercalated cells, are able to respond to vasopressin. On the cellular level, the cytoplasmic staining of collecting tubule cells might reflect endocytosed or sequestered vasopressin receptor proteins. The apical staining of some cells of the tubular epithelium, observed in this study and also reported by Ravid et al. [37], seems to be in contrast with earlier functional assays which localize the CAMP-coupled response of the V,-receptor to the basolateral membrane [43]. Recent studies, however, suggest that luminal vasopressin modulates electrogenie ion transport in cortical collecting ducts [44] and water urea transport in inner medullary collecting ducts [45]. Our staining patterns provide further evidence that vasopressin receptors occur in locations other than the basolateral membrane and that luminal (urinary) vasopressin could interact with vasopressin receptors on the apical cell membrane. In addition to V,-receptors, V,-receptors may be responsible for staining of the principal cells as shown for the rabbit collecting tubule [46]. The staining of glomerular structures and blood vessels probably reflects Vi-receptors [47], which is again consistent with recognition of both vasopressin receptor subtypes by the antiidiotypic monoclonals. In summary, this study describes the production and properties of anti-idiotypic antibodies to vasopressin receptors and their use for immunolocalization of vasopressin receptors in the kidney, a major target organ for vasopressin. These antibodies offer interestingpossibilities for the study of vasopressin receptor expression by light and electron microscopy in different target organs, including the central nervous system. Immunohistochemical studies of the rat brain using these monoclonal antibodies are currently in progress. Patricia Jans is gratefully acknowledged for expert assistance with cell cultures. Dr. B. A. Hemmings (CIBA-Geigy, Basel) and Dr. H. Luzius (MPI fur Biophysik, Frankfurt) are thanked for making the Ml8 and VPRl cell lines available. We are grateful to Dr. H. J. Knot (Sandoz, Basel) for the IP, determination. An essential part of this work belongs to the Ph.D. thesis of M.J. (Frankfurt University). This work was supported by the Deutsche Forschungsgemeinschaft (SFB 169 and SFB 177).

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