ELSEVIER Journal of Immunological Methods 2 I2 ( 1998) 9-18

JOUIINAL OF IM~ICAL METtUHJS

Detection of biotinylated cell surface receptors and MHC molecules in a capture ELISA: a rapid assay to

measure endocytosis

Diane N. Turvy, Janice S. Blum *

Department of Microbiology and Immunology. Indiana lJniaersi@ School of Medicine. Indianapolis. IN 46202-5120. USA

Walther Cancer Institute, Indianapolis, IN 46202-5120. USA

Received I I August 1997: accepted 25 November 1997

Abstract

Cell surface receptors and antigens, such as TW and MHC molecules, are endocytosed and subsequently redisplayed on the plasma membrane. The internalization and recycling of MHC molecules is thought to play an important role in antigen

presentation, but studying this process has been hindered due to the lack of a rapid and easily quantitated assay. The combination of a cleavable biotin reagent to label surface molecules and a capture ELISA to detect these molecules of interest, allows for the quantitation of their cell surface expression, endocytosis and recycling. The endocytosis of TW and

MHC II molecules was readily quantitated in B cell lines using this procedure with results nearly identical to previously published data using more laborious radioactive methods. Evidence for the recycling of class II antigens and TfR back to the plasma membrane was obtained by monitoring the exit of these molecules from endosomes. Exposing cells to hypertonic media blocks clathrin-dependent endocytosis and was found to inhibit the internalization of MHC class II proteins on B

cells. This flexible assay to capture and quantitate the cell surface expression and endocytosis of MHC molecules and other surface antigens offers a sensitive and non-radioactive alternative to study the intracellular trafficking of diverse membrane

proteins. 0 1998 Elsevier Science B.V. All rights reserved.

Keywords: Biotinylation; MHC; TfR; Endocytosis: APC

Abbreviations: APC, antigen presenting cells: TW, transferrin receptor; MHC, major histocompatibility complex: NHS, N-hydroxy-

succinimide; HRP, horseradish peroxidase; TAP, transporter for antigenic peptides: HBSS, Hanks balanced salt solution: ELISA, enzyme

linked immunosorbent assay; GSH. glutathione; PBS, Dulbecco’s phosphate-buffered saline; PBS-T, PBS plus Tween 20; Pq, Primaquine;

SDS-PAGE, sodium dodecyl sulfate-polyacrylamide gel electrophoresis; BSA, bovine serum albumin; ECL, enhanced chemiluminescence:

FcR, IgG receptor; PMSF. phenylmethylsulfonyl fluoride; TLCK, No-p-tosyl-L-lysine chloro-methyl ketone

* Corresponding author. Department of Microbiology and Immunology, Medical Science Bldg., Rm. 255, Indiana University, 635

Barnhill Drive, Indianapolis, IN 46202.5120, USA. Tel.: + l-3 17-278-1715; fax: + I-317-274-4090: e-mail: jblum@indyvax.iupui.edu.

0022-1759/98/$19.00 0 1998 Elsevier Science B.V. All rights reserved.

PII SOO22- 1759(97)00206-8

10 D.N. Turry. J.S. Blum/Journal of Immunolo@zl Methods 212 (1998) 9-18

1. Introduction

The endocytic pathway functions as a key gate- way delivering nutrients, macromolecules and pathogens into immune cells. B lymphocytes,

macrophages and dendritic cells function as antigen presenting cells with enhanced capacities for trans- porting molecules from the cell surface into endoso-

ma1 vesicles (Brodsky and Guagliardi, 1991). A variety of cell surface receptors as well as major

histocompatibility (MHC) antigens have been shown to enter the endocytic pathway of APC. Extensive

studies have demonstrated the rapid endocytosis and recycling of transferrin receptors (TW) (Ajioka and

Kaplan, 1986). TfR is endocytosed into clathrin

coated pits, which pinch off from the plasma mem- brane and form clatbrin coated vesicles. The vesicles rapidly uncoat and fuse together or with pre-existing

vesicular structures called early endosomes, where removal of a ligand may occur. The molecules then

return to the plasma membranes, possibly via carrier vesicles (Gruenberg and Howell, 1989; Pearse and

Robinson, 1990). More recently, MHC class I and 11 antigens have been shown to endocytose and recycle

although far less is known about this process and its regulation (Tse and Pemis, 1984; Reid and Watts,

1990). The recycling of MHC antigens is thought to play an important role in antigen presentation. The

release of unstable peptides from MHC antigens may be facilitated upon transport through acidic periph- eral endocytic compartments containing proteases. Thus, it has been suggested that MHC antigen recy- cling through endosomes may favor presentation of peptides with the highest affinity (Motal et al., 1993).

Assays to measure the endocytosis and recycling of MHC antigens are currently cumbersome involv-

ing iodinated antibodies, cleavable radiolabelling

reagents (Bretscher and Lutter, 1988) or biotinylated F(ab) fragments detected with radiolabeled avidin (Pinet et al., 1995). Biotin-labeling of cell surface antigens has been established as a methodology for monitoring transport to the cell surface (Brachet et al., 19971, polarized sorting (Marmorstein et al., 1996) and endocytosis (Volz et al., 1995). For recep- tors such as TfR and MHC class II antigens, the addition of a biotin tag has been shown not to perturb surface expression or endocytic transport (Volz et al., 1995; Pinet et al., 1995). Yet, direct quantitation of the cell surface and endocytosed bi-

otin-tagged proteins has been cumbersome and relied

upon PAGE followed by densitometry. Here the techniques of protein biotinylation in viable cells has been extended to permit direct, accurate quantitation of surface expression, endocytosis and recycling for a wide variety of plasma membrane proteins includ-

ing receptors and MHC antigens. Sulfo-N-hydroxy- succinimide-.S-Sbiotin is used to chemically label cell surface proteins on APC. Biotinylation of sur-

face proteins provides a rapid and safe alternative to radioactive labeling that is independent of ligand

binding. N-hydroxysuccinimide (NHS) is a highly reactive ester group which introduces a biotin tag

covalently into any polypeptide that contains either an unblocked NH,-terminal amino acid or an ex-

posed amino group of a reactive lysine residue (Anderson et al., 1964). Since lysine residues ac- count for 7% of the total amino acid residues in an average protein, the probability that biotinylation of any single protein will occur is extremely high (Luna,

1996). The Sulfo-NHS-S-S-Biotin is advantageous due to its water solubility and membrane imperme-

ability, thus allowing only cell surface proteins to be labeled. Endocytosis of the biotin-tagged proteins

can be detected following incubation of cells at 37°C. Treatment of these cells with a reducing reagent releases biotin from cell surface proteins, while en-

docytosed molecules are protected and retain their biotin label. The amount of biotin bound to a specific receptor or antigen can be quantitated using an anti- body capture technique and avidin horseradish per- oxidase (HRP). Extensive studies with this assay demonstrated that the basal levels of TfR or MHC

class II antigen endocytosis measured were compara- ble to results obtained with more complex radioac- tive assays. Biotin labeling was specific for cell

surface proteins, and this modification did not pro- mote the endocytosis of proteins lacking intemaliza- tion signals such as the B-lymphocyte Fc receptor

(Amigorena et al., 1992). Furthermore, the assay allows rapid comparison of the endocytosis of a wide variety of cell surface molecules on immune cells.

2. Materials and methods

2.1. Cell line

T2.DR4 was generated by stable transfection of MHC class II DR4w4 (Y p subunits into the class II

D.N. Tuny, J.S. Blum/ Journal of Immunological Methods 212 (1998) 9-18 II

null T2 cell line (Salter et al., 1985). The T2 parent

cell is a hybrid of the human B cell, 72 1.174 and the human T cell, CEM, which lacks all four copies of

the MHC class II region of chromosome 6. T2 has defects in the transporter for antigenic peptides (TAP proteins), which result in an inability to load endoge-

nous peptides into class I molecules. Failure of MHC class 1 antigens to associate with a peptide renders these molecules unstable and therefore surface ex- pression of class I proteins is greatly reduced in T2 cells.

2.2. Biotinylation of su$ace proteins

Sulfosuccinimidyl-2-(biotinamido) ethyl-1,3-di-

thiopropionate (Sulfa-NHS-S-SBiotin; Pierce) was dissolved in HBSS at 10 mg/ml. Sulfo-NHS-SS-

Biotin was added to 3 X lo7 cells in HBSS at a concentration of 0.5 mg/ml. Cells were rotated at 4°C for 1.5 min for biotin surface labeling followed by three washes with cold HBSS plus 5 mM Tris pH 7.4 to quench any excess NHS. Cells were then lysed to permit quantitation of surface biotinylated pro-

teins. To determine the total amount of a specific protein within cells using this biotin reagent, an

aliquot of cells was lysed prior to biotinylation, and then the excess biotin was quenched by adding 5

mM Tris pH 7.4. Biotin labeled proteins were quanti- tated using an antibody capture ELISA.

2.3. Endocytosis

Biotinylated cells were incubated at 37°C for 30

min in a CO, incubator (ambient CO, 7.0%) to facilitate endocytosis or internalization of surface receptors and antigens. The kinetics of MHC class II

endocytosis was determined by incubating biotinyl- ated cells at 37°C for varying lengths time prior to glutathione stripping. Endocytosis was blocked by treating cells with 0.45 M sucrose in HBSS for 10 min at 37°C prior to biotinylation with an additional sucrose treatment during the 30 min incubation at 37°C following biotinylation (Heuser and Anderson, 1989). In contrast with sucrose treatment, pri- maquine allows cell surface endocytosis, but blocks the cycling of proteins back to the plasma membrane (Schwartz et al., 1984; Stoorvogel et al., 1996). Cells were treated with 0.3 mM primaquine in RPMI-

Hepes during the 30 min incubation at 37°C to allow

endocytosis without recycling. Control studies to monitor trypan blue exclusion by viable cells demon- strated that neither sucrose nor primaquine treatment

was toxic to the cells during a 30 min incubation.

2.4. Glutathione stripping of biotin

To release the biotin label from proteins at the cell surface, cold glutathione solution (Bretscher and

Lutter, 1988) was added to the cells at 4°C. The solution was made as follows: 0.05 mM glutathione, 0.075 mM NaCl, 0.001 mM EDTA were dissolved

in water and just prior to use 0.075 mM NaOH and then 10% of fetal calf serum was added. Cells were rocked with the GSH solution for 20 min at 4°C

followed by pelleting and resuspension in fresh cold glutathione solution with rocking for 30 min at 4°C. The cells were then washed three times with HBSS prior to lysing. To demonstrate the continuous cy- cling of endocytosed proteins back to the cell sur- face, glutathione stripped cells were rewarmed to 37°C for 30 min to permit endocytosis, followed by a

second glutathione treatment. The additional glu- tathione treatment removed biotin from any biotinyl-

ated receptors or antigens that recycled back to the plasma membrane during the second warming. This

process of warming and glutathione stripping cells multiple times, allows measurement of protein recy- cling from endosomes to the plasma membrane.

2.5. Cell lysates

Cells prior to or following biotin-labeling, were

lysed at 4°C in HBSS with 1% Triton X-100 and the protease inhibitors PMSF (0.02 mM) and TLCK

(0.01 mM). Lysed cells were incubated on ice for 15 min and then spun at 1000 rpm for 5 min at 4°C to remove the intact nuclei. As a control for Western

immunoblots, lysates were also generated from cells without biotin-labeling.

2.6. ELISA to detect biotinylated proteins

An antibody capture ELISA was used to quanti- tate receptor or antigen surface expression and endo- cytosis. COSTAR EIA/RIA 96-well flat bottom

plates were coated overnight at 4°C with 100 ~1 of

12 D.N. Turry. J.S. Blum/.Journal of Immunological Methods 212 (1998) 9-18

antibody specific for the antigen being assayed. Specifically, 37.1 (anti-human MHC II, provided by L. Wicker, Merck, Rahway, NJ) was used at 5 pg/ml; B3/25 (anti-human transferrin receptor, generously provided by Dr. Ian Trowbridge) was used at 5 pg/ml; Cdw32 (anti-human FcR. PharMingen) was used at 1 Fg/ml; W6/32 (anti-

human MHC I, ATCC) was used at 5 ,cLg/ml; along with the sera X-13 (rabbit anti-human cathepsin D,

Blum et al., 1991). Plates were washed three times with 300 pi/well of PBS plus 0.05% Tween-20

(PBS-T). Each plate was then blocked by adding 300 pi/well of PBS plus 5% of fetal calf serum three times, allowing each addition to incubate for 10 min at room temperature. Following three additional washes with PBS-T, cell lysates (100 pi/well) were added to the plate and incubated for 2 h at 4°C.

As controls, cell lysis buffer was added to antibody coated wells or alternatively, cell lysates were added to wells coated with fetal calf serum. The plate was

washed five more times with PBS-T. Avidin-per-

oxidase (2.5 pg/ml) in PBS plus 10% calf serum was added (100 pi/well) and then incubated at

room temperature for 30 min, followed by eight washes with PBS-T. Freshly prepared ABTS (Sigma) +O.l% hydrogen peroxide was used to develop the plate, with color being read at 405 nm. The results given are representative of experiments that were repeated at least three times. with triplicate data obtained from each individual experiment and used

to calculate standard deviations in each measure- ment. The absorbance values were compared using

one way analysis of variance (ANOVA). The Tukey HSD procedure was used to define differences when the ANOVA was significant. Before running the analysis the adjusted percentage was calculated by

subtracting the average antibody control background from all values including test samples, total cell or cell surface label for each protein of interest. These corrected sample values were then divided by either the corrected average total cell labeling or by the corrected average cell surface label for each protein. followed by multiplication by 100 to yield the ad- justed percentage of protein biotinylation.

2.7. Western blot

As an additional means of detecting class II anti- gens tagged with biotin, Western blots were carried

out. The biotin-labeled cell lysates were immunopre- cipitated with 37.1 at 5 pg/ml under conditions similar to the antibody capture ELISA. These com- plexes were bound to protein A sepharose beads pre-coated with rabbit anti-mouse IgG. Antigen-an-

tibody complexes associated with beads were washed several times with Tris saline (10 mM Tris pH 7.4,

150 mM NaCl, 0.1% Triton X- 100). The precipitates were eluted with reducing SDS PAGE sample buffer,

separated on a 10% SDS polyacrylamide gel, fol- lowed by transfer to nitrocellulose. As a control, a

non-biotin-labeled cell lysate was electrophoresed and transferred with biotin-labeled samples. After blocking the nitrocellulose overnight in PBS-T plus

1% BSA, the blot was cut and the biotin samples were probed with streptavidin conjugated to horseradish peroxidase (1 pg/ml) for 30 min at 4°C. The unlabeled control sample was probed with the antibodies XD5.All (specific for MHC II /3 chain)

and DA6.147 (specific for MHC II (Y chain) for 2 h at 4”C, followed by incubation with goat anti-mouse

IgG conjugated to horseradish peroxidase for 30 min

at room temperature. The blots were then developed with enhanced chemiluminescence (ECL) according to the manufacturer’s instructions (Amersham) and exposed to X-ray film.

3. Results and discussion

3.1. Endocytosis of biotinylated proteins

Select proteins on the surface of cells are sorted

into clathrin coated pits and delivered into the endo- cytic pathway. Studies of this transport process for plasma membrane receptors and antigens has been dependent upon the availability of radiolabeled lig- ands or affinity reagents for individual proteins (Bretscher and Lutter, 1988; Pinet et al., 1995). To facilitate studies of MHC antigens and receptors on APC, a non-radioactive methodology was devised to quantitate surface expression and endocytosis. As

indicated in Fig. 1, this assay detects even low levels of endocytosis for specific cell surface molecules, but does not induce aberrant internalization of pro- teins from the plasma membrane. Thus, the endocy- tosis of TfR and MHC class II antigens was readily

13

120

100

FcR

Cell Surface Molecule

Catheprin D MHC I

I 8Total Cell

@Cell Surface ’

R#GSH Snipped

1 m Endocytosis

Fig. 1. Biotinylated cell surface molecules can be detected using a capture ELISA. The ceil surface level of each molecule was measured by

biotinylation of intact cells. A glutathione solution removed the surface biotin by disulfide cleavage either immediately after biotinylation or

after a 30 min incubation 37°C to permit endocytosis. To determine the total cellular expression of each protein. cells were biotinylated after

lysis. Proteins were captured on ELISA plates coated with antibodies specific for each molecule tested. TfR and MHC II are both known to

recycle while FcR and cathepsin D serve as negative controls. FcR is a cell surface molecule that does not endocytose in B cells and

cathepsin D is strictly found intracellularly, confirming the membrane impermeability of the biotin-label. T2.DR4 are defective in TAP and

therefore do not express significant levels of MHC class 1 on the cell surface confirming the specificity of the assay for surface proteins. The

percent protein biotinylated was calculated from the ELISA data, which is expressed as absorbancrs at 405 nm from a representative

experiment with triplicate samples and corrected for non-specific binding. according to the materials and methods. Briefly. the corrected

absorbance for each sample was divided by the corrected absorbance for the total cellular content of each specific protein followed by

multiplying by 100 to yield the percent protein biotinylation.

detectable in contrast with the FcR which lacks the from the cell surface in 30 min; a figure comparable appropriate signal sequences for internalization in B to published estimates of 60% TW endocytosis using cells (Amigorena et al., 19921. For these experi- other assay systems (Bretscher and Lutter, 1988). ments, sulfo-NHS-S-S-biotin was used to label pro- Within the same time span, the endocytosis of MHC teins on the surface of viable T2.DR4 cells. Follow- class II proteins was far less efficient with only ing incubation of these labeled cells at 4°C or 37°C 15.6% of these molecules transiting to endosomes. the cells were treated with a glutathione solution to Again, these results are comparable to the reported release residual biotin from molecules remaining on 15-20% of MHC 11 reaching endosomes in B cells the cell surface. As expected, incubation of cells at as determined using radioactive assays (Reid and 4°C blocked protein endocytosis. Studies at 4°C indi- Watts, 1990; Pinet et al., 19951. Similar levels of cated between 95-100% of the biotin label was MHC class II and TfR endocytosis were detected in

removed from surface proteins using a glutathione experiments with multiple EBV transformed B cell solution. By contrast at 37”C, our assays demon- lines. Confirmation that the biotin label tags only cell

strated approximately 59% of the TfR endocytose surface proteins was obtained via studies of an endo-

14 D.N. Turvy. J.S. Blum/Joumal of Immunological Methods 212 (1998) 9-18

somal/lysosomal protease, cathepsin D. This intra- cellular protease could be readily biotin-labeled in detergent lysed cells, but was not tagged by cell surface biotinylation. In addition, we took advantage of a unique property of the T2.DR4 cell line as an additional control. MHC I, although typically abun-

dant on cell surfaces, is not displayed efficiently on T2.DR4 cells due to a defect in TAP, the transporter

for antigenic peptides. Thus similar to cathepsin D,

MHC I proteins found within T2.DR4 could be biotin-labeled upon cell lysis, but few class I proteins were detectable on the cell surface. The recycling of

MHC I proteins has been reported in T lymphocytes

(Metal et al., 19931, however we were unable to detect this process in T2.DR4 cells due to the low level of surface class I molecules. Studies using this biotin assay and another B-LCL expressing normal levels of MHC I indicated minimal endocytosis, 3%,

of these antigens (M. Andis, unpublished data) in

keeping with published results in B cells (Tse and Pemis, 1984).

3.2. Recycling from endosomes to the cell surface

To demonstrate recycling of internalized biotin-

labeled molecules from endosomes back to the

,-

I..

,..

I..

Fig. 2. Detection of TFR and MHC II antigen endocytosis and recycling. To demonstrate recycling of biotin labeled proteins from the cell

surface through endosomes and back to the plasma membrane, cells were subjected to multiple rounds of warming and GSH stripping. Two

rounds of warming for 30 min at 37°C to allow endocytosis followed each time by glutathione cleavage, confirmed that an individual

molecule can internalize and recycle to the cell surface acquiring glutathione sensitivity again. Primaquine (Pq) permits endocytosis but

blocks recycling of molecules to the cell surface thus resulting in increased amounts of intracellular biotinylated MHC class II and TW.

Primaquine was added to intact cells during the 30 min incubation at 37°C. Sucrose treatment of cells inhibited the endocytosis of MHC I1

completely, but only 77% of TfR endocytosis was blocked. Biotinylated cells were incubated at 37°C for 30 min in the presence or absence

of 0.45 M sucrose. The cell surface expression and endocytosis of proteins was determined as described in Fig. 1. The biotinylated cell surface molecules were detected in a capture ELISA yielding triplicate data. The percent biotinylated protein was calculated as an adjusted

percent as described in materials and methods with the cell surface amount of each protein being set at 100%.

Tab

le

1 D

etec

tion

of T

fR

and

MH

C

II e

ndoc

ytos

is

and

recy

clin

g

TfR

Mea

n

abso

rban

ce

405

nm

Stan

dard

A

djus

ted

devi

atio

n (a

)

Stan

dard

devi

atio

n

MH

C

II

Mea

n

abso

rban

ce

405

nm

Stan

dard

devi

atio

n

Adj

uste

d

(%I

Stan

dard

devi

atio

n

Cel

l su

rfac

e 1.

064

0.01

7 10

0 1.

103

0.02

10

0 G

SH

stri

pped

0.

083

0.00

7 1.

8 0.

7 0.

115

0.00

3 4.

7 0.

3 In

tern

aliz

ed

0.69

4 0.

012

59.3

1.

2 0.

235

0.00

7 15

.6

0.6

Inte

rnal

ized

pl

us

Pq

0.76

6 0.

015

66.1

1.

4 0.

279

0.00

9 19

.6

0.8

Rec

yclin

g (t

wo

roun

ds

of w

arm

th

en

stri

p)

0.45

0.

018

36.3

1.

6 0.

201

0.01

1 12

.5

1 E

ndoc

ytos

is

plus

su

cros

e 0.

211

0.00

2 13

.9

0.1

0.13

7 0.

005

6.7

0.4

Ant

ibod

y co

ntro

l 0.

063

0.00

1 0.

063

0.00

3

“Cal

cula

ted

acco

rdin

g to

mat

eria

ls

and

met

hods

.

Mea

n ab

sorb

ance

va

lues

ar

e fr

om

trip

licat

e da

ta

with

th

e st

anda

rd

devi

atio

n of

ea

ch

trip

licat

e gi

ven.

T

he

adju

sted

pe

rcen

t pr

otei

n bi

otin

ylat

ed

and

its s

tand

ard

devi

atio

n w

as

calc

ulat

ed

acco

rdin

g IO

the

mat

eria

ls

and

met

hods

w

ith

the

cell

surf

ace

amou

nt

of

each

pr

otei

n be

ing

set

to

100%

. T

he

diff

eren

ce

betw

een

each

sa

mpl

e w

as

dete

rmin

ed

to b

e

sign

ific

ant

by

stat

istic

al

anal

ysis

.

16 D.N. Turcy, J.S. Blum /Journul qf Immunological Methods 212 (lYY8,8) Y-18

plasma membrane, the cells were permitted to endo-

cytose labeled proteins and the remaining surface biotin was released by glutathione cleavage. These

cells were then warmed to permit re-expression of the internalized biotin-labeled molecules. GSH treat-

ment of cells exposed to multiple cycles of warming, resulted in a significant loss of biotin label from

proteins such as TfR and MHC II as they cycled

back from endosomes to the cell surface (Fig. 2, Table 1). Thus, there was a greater reduction in the amount of biotinylated protein in the cells subjected to two rounds of warming and glutathione treatment compared to those which only underwent a single

round of this process. Our results indicate a 20% reduction in MHC II labeled and a 40% reduction in transferrin receptor biotinylation when recycling of

these molecules was allowed to occur at 37°C. As an

additional probe to establish recycling of the biotin- labeled proteins, cells were treated with primaquine

which non-specifically disrupts endosomal/lyso- somal function. Primaquine has been shown to slow

the rate of recycling of TfR (Schwartz et al., 1984; Stoorvogel et al., 1996) and MHC II (Reid and Watts, 19901, and thereby should increase the amount of biotin-labeled proteins within endosomes using our assay. With the addition of primaquine the pro- portion of labeled TfR and MHC II molecules that

are resistant to glutathione increased by 11.6% and 25.6% respectively. These increases were determined

to be significant by statistical analysis with both yielding p values of less than 0.001. The mecha-

nism by which primaquine blocks receptor recycling has not been fully elucidated, but most likely in-

volves dissipation of endosomal pH gradients and osmolarity.

3.3. Hypertonicity blocks endocytosis

Exposure of cells to hypertonic media blocks

receptor mediated endocytosis by preventing the for- mation of clathrin coated pits (Daukas and Zigmond,

1985). Microscopy studies have revealed that hyper- tonic media has the same effect as K+ depletion in

triggering cells to display empty clathrin ‘micro- cages’ at the cell surface (Larkin et al., 1983). Abnormal formation of microcages inhibits endocy- tosis by rendering clathrin unavailable for assembly into normal coated pits (Heuser and Anderson, 1989).

The addition of hypertonic sucrose solutions reduced

the endocytosis of MHC class II proteins to back- ground level, confirming that endocytosis of MHC II

is mediated by clathrin coated pits (Fig. 2, Table 1). Truncation of the cytoplasmic tails of MHC class II

or TfR has been shown to inhibit internalization of the molecules suggesting that an interaction of the

tails with a protein in the coated pits is essential for

endocytosis (Pinet et al., 1995; Rothenberger et al., 1988). The proportion of intracellular biotin-labeled

TfR was also decreased by hypertonic treatment of cells, although a measurable number of these recep- tors were observed to still endocytose. This results

confirm that endocytosis of TfR is mediated by both clathrin coated pits and mechanisms that are inde-

pendent of clathrin (Bos et al.. 19951.

3.4. Kinetics of MHC class II endocytosis

The kinetics of MHC class II endocytosis in

T2.DR4 cells was measured over a one hour time period. The endocytosis of these molecules began almost immediately after warming, as seen in Fig. 3.

The endocytosis continued to increase during the first 30 min and then levelled off at approximately 15%. This probably represents a balance between additional endocytosis of biotin-labeled MHC class

Fig. 3. Kinetics of MHC class II endocytosis. The rate of endocy-

tosis of MHC class II molecules was examined by varying the

length of incubation at 37°C. Biotinylated cells were glutathione

stripped after warming to 37°C for 0 to 60 min. The cell surface

expression was determined as described in Fig. I and used to calculate the percent of endocytosis for each sample. The data is

calculated from triplicate samples of a representative experiment.

D.N. Turry. J.S. Blum/.Journal oflmmunolo,~ical Methods 212 f 1998) 9-18 17

II antigens and the recycling of these biotinylated

antigens to the cell surface where they become sus- ceptible to glutathione treatment.

3.5. Recycling of A4HC II does not perturb the dimeric structure of MHC II antigens

Early endosomal vesicles of APC are mildly acidic

(pH 6.51 and contain active cysteine and aspartyl

proteases (Diment and Stahl, 198.5). To determine if transit through these organelles resulted in proteoly-

sis of MHC II. the structure of the class II LY and p subunits were assessed by Western blotting (Fig. 4). The molecular mass of each class II subunit was very slightly increased upon biotin labeling of these

proteins on the cell surface (compare lane 1 and 21 compared with native MHC II detected using anti- bodies to DR LY and p. Endocytosis of the biotin- labeled class II proteins did not alter their elec-

trophoretic mobility or dimeric structure (lane 4). For

Fig. 3. Western blot analysis demonstrated the structural integrity

of class II c~ and p dimers after biotinylation and endocytosis. To

demonstrate the electrophoretic mobility of native MHC II. cell

lysate from T2.DR4 was electrophoresed. transferred and probed

with XD5.A 1 I (anti-human MHC class II beta chain) and DA6.147

(anti-human MHC class II alpha chain) followed by detection with

goat anti-moue IgG conjugated to HRP (lane I). Lane 2 is cell

surface hiotin-labeled MHC class II molecules: lane 3 shows the

removal of biotin from cell surface MHC II by GSH stripping;

lane 3 is endocytosed MHC II (30 min, 37°C) after surface GSH

stripping: and in lane 5, cells were incubated in hypertonic sucrose

for 30 min at 37°C followed by GSH stripping. confirming a lack

of MHC II endocytosis under these conditions. In lanes 2-5,

immunoprecipitated MHC II was isolated with the 37.1 antibody

and protein A sepharose coated with rabbit anti-mouse IgG. The

immobilized MHC II molecules were eluted from the beads with

SDS reducing sample buffer and electrophoresed. After transfer-

ring to nitrocellulose, the biotinylated class II molecules were

detected by probing with streptavidin conjugated to HRP. ECL

was used to visualize the probed molecules. The position of MHC

II a and p is indicated on the right.

these experiments, MHC II was biotin labeled and captured with the 37.1 antibody which recognizes a conserved region of the (Y 2 p 2 domain of HLA-DR molecules. Dimerization of class II (Y and p is dependent upon binding of peptides by these MHC proteins, thus the recycling proteins captured by these assays presumably contain their peptide lig-

ands. For these blots, the antibody-captured class II antigens were eluted with PAGE sample buffer, ana-

lyzed by Western blotting and detected using strepta- vidin linked to horseradish peroxidase. As observed,

this method immunoprecipitated only class II pro- teins and confirmed the specificity of our capture ELISA. Both immunoblotting and the ELISA are

comparably sensitive for detecting biotin-labeled proteins and endocytosis. The ELISA method offers the added advantage of rapid quantitation of even low levels of protein/receptor endocytosis and recy-

cling. Recent studies have suggested that early endo-

somes may function as a site of antigen processing and/or peptide loading for MHC class II proteins

(Jensen, 1990). There is some evidence that class I1 molecules may enter this endosomal compartment to acquire or exchange peptides (Reid and Watts, 1992).

Further studies of the importance of endocytic trans- port and recycling are needed for both MHC class I and class II antigens. Thus, the sensitive and easily adaptable assay described here, should be of use to

these important investigations.

Acknowledgements

The authors wish to thank Melissa Andis, Jill

Beitz and John Lich for their technical assistance. This work was supported by the NIH grants A13341 8 and DK9401T. Statistical support was provided by Dr. Naomi Fineberg through the NIH-funded Dia- betes Research and Training Center in Indiana.

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