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IMMUNOBIOLOGY
IgG plasma cells display a unique spectrum of leukocyte
adhesion and homing molecules
Gregory H. Underhill, Heather A. Minges-Wols, Jamie L. Fornek, Pamela L. Witte, and Geoffrey S. Kansas
Long-lived antibody-secreting plasma
cells are formed in the secondary lymphoid organs and subsequently home to
the bone marrow, although the mechanisms that control this migration remain
primarily unknown. In this study, we show
that IgG plasma cells constitute a significant fraction of cervical lymph node cells
from older mice deficient in both E- and
P-selectin (E/Pⴚ/ⴚ), and that these cells
can be prospectively isolated by phenotype. These IgG plasma cells were polyclonal, cytoplasmic Igⴙ, spontaneously
secreted antibody, were in the G0/G1 phase
of the cell cycle, and failed to express
multiple B-cell surface markers. The
plasma cells exhibited up-regulated cell
surface expression of multiple adhesion
molecules, including ␣4 and leukocyte
function-associated antigen 1 (LFA-1) integrins, CD44, and P-selectin glycoprotein ligand 1 (PSGL-1). IgG plasma cells
bound to vascular cell adhesion molecule 1
(VCAM-1) significantly better than IgMⴙ B
cells, indicating that the ␣4 integrins were
constitutively active. A subset of IgG plasma
cells also bound hyaluronic acid, the ligand
for CD44. In addition, the IgG plasma cells
interacted strongly with E-selectin, but
poorly with P-selectin, despite elevated
levels of PSGL-1 protein. The preferential
interaction of plasma cells with E-selectin,
but not P-selectin, correlated with elevated
␣1,3-fucosyltransferase-VII messenger RNA
levels, but selective down-regulation of core
2 ␤1-6-N-glucosaminyltransferase levels,
compared to B cells. These results demonstrate a unique adhesion profile for murine
IgG plasma cells. Furthermore, the E/Pⴚ/ⴚ
mice represent a novel system to isolate
and purify significant numbers of primary
IgG plasma cells. (Blood. 2002;99:2905-2912)
© 2002 by The American Society of Hematology
Introduction
Long-lived plasma cells have recently been identified as an
important component of B-cell memory,1,2 and the majority of
long-lived plasma cells are localized in the bone marrow.2,3
Migration of newly formed plasma cells from the lymph nodes and
spleen to bone marrow is therefore an important step in the
maintenance of a long-term antibody response to a pathogen.
Adoptive transfer of bone marrow containing plasma cells from
mice 4 months after lymphocytic choriomeningitis virus (LCMV)
infection into naive recipients results in significant long-term
LCMV-specific serum IgG levels, suggesting that long-lived plasma
cells may maintain their capacity to home to bone marrow.1 Plasma
cells have also been identified at sites of chronic inflammation in
diseases such as rheumatoid arthritis.4 It is unknown to what extent
plasma cells are generated locally in these inflammatory tissues, or
if plasma cells are formed in lymphoid organs and home to these
sites. Thus, although the formation of plasma cells in an immune
response has been extensively studied, little is known about the
molecular mechanisms and adhesion molecules that govern normal
plasma cell migration.
The selectins are a family of carbohydrate-binding adhesion
molecules that mediate the earliest steps of leukocyte interaction
with the vessel wall.5 E-selectin and P-selectin are members of the
selectin family that are inducibly expressed in most tissues during
an inflammatory response, but are also constitutively expressed in a
limited number of tissues.5 For example, E-selectin is constitu-
tively expressed in human and murine bone marrow.6,7 Bone
marrow endothelial cells support rolling of fetal liver hematopoietic progenitor cells (HPCs) and an HPC-like cell line.8 This rolling
was mediated by both E-selectin and P-selectin and vascular cell
adhesion molecule 1 (VCAM-1). E-selectin, P-selectin, and ␣4/
VCAM-1 interactions were also found to be important in homing of
HPCs to the bone marrow after irradiation and transplantation.9
Furthermore, ␣4-null chimeric mice demonstrate that ␣4 integrins
play a role in the attachment and transmigration of pre-B cell lines
beneath bone marrow stroma, and these chimeric mice also display
reduced myeloid and B-lymphoid progenitor numbers in the bone
marrow.10 Whether similar mechanisms control the migration of
plasma cells to bone marrow is unknown.
E-selectin and P-selectin double-deficient (E/P⫺/⫺) mice
spontaneously develop mucocutaneous infections and exhibit a
severe reduction in leukocyte rolling, strongly elevated blood
leukocyte counts, and an absence of early neutrophil migration
in induced peritonitis.7,11 E/P⫺/⫺ mice also display severe
cervical lymphadenopathy, including expanded numbers of
lymphocytes, macrophages, and most notably, plasma cells.11 In
addition, serum IgG levels are increased about 10-fold.11 In this
report, we show that E/P⫺/⫺ mice represent a novel source from
which to purify significant numbers of primary IgG plasma cells
and demonstrate that these cells exhibit a unique constellation of
adhesion receptors.
From the Department of Biomedical Engineering, Northwestern University,
Evanston, IL; Departments of Microbiology-Immunology and Cell Biology,
Loyola University Health Science Center, Maywood, IL; and Department of
Microbiology-Immunology, Northwestern Medical School, Chicago, IL.
Reprints: Geoffrey S. Kansas, Department of Microbiology-Immunology,
Northwestern Medical School, 303 E Chicago Ave, Chicago, IL 60611; e-mail:
[email protected].
Submitted September 20, 2001; accepted December 3, 2001.
Supported by NIH grants HL58710 (to G.S.K.) and AG13874 and K07
AG00997 (to P.L.W.). G.S.K. was an Established Investigator of the American
Heart Association.
BLOOD, 15 APRIL 2002 䡠 VOLUME 99, NUMBER 8
The publication costs of this article were defrayed in part by page charge
payment. Therefore, and solely to indicate this fact, this article is hereby
marked ‘‘advertisement’’ in accordance with 18 U.S.C. section 1734.
© 2002 by The American Society of Hematology
2905
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2906
BLOOD, 15 APRIL 2002 䡠 VOLUME 99, NUMBER 8
UNDERHILL et al
Materials and methods
Mice
The E-selectin/P-selectin double-deficient mice,11 backcrossed 5 generations to C57BL/6, were provided by Dr Dan Bullard (University of
Alabama-Birmingham). Wild-type C57BL/6 mice were purchased from
Jackson Laboratories (Bar Harbor, ME) and bred in our mouse facility.
E/P⫺/⫺ mice were generally 3 to 12 months of age when used.
EDTA, with protease inhibitors. Rabbit antimouse Blimp-1 antiserum was
kindly provided by Dr Mark Davis (Stanford University, Stanford, CA) and
rabbit antimouse BCL-6 was purchased from Santa Cruz Biotechnology
(Santa Cruz, CA). Horseradish-peroxidase–conjugated antirabbit Ig second
step was purchased from Biosource and nitrocellulose membranes were
developed using enhanced chemiluminescence reagents (Amersham Pharmacia Biotech, Piscataway, NJ). The murine plasmacytoma cell line, J558,
was used as a positive control for Blimp-1 expression, and BJAB cells were
used as a positive control for BCL-6.
ELISPOT analysis
Cell isolation
For isolation of the B-cell compartment by depletion of T cells and myeloid
cells, E/P⫺/⫺ or wild-type cervical lymph node cells were incubated for 15
minutes at 4°C with anti-CD5 and anti-CD11b microbeads (Miltenyi
Biotec, Auburn, CA). Cells were then passed through a CS depletion
column (Miltenyi Biotec) according to the manufacturer’s protocol. For
plasma cell isolation, cervical lymph node cells were incubated with
anti-CD5, anti-Mac-1, anti-IgM, and anti-B220 microbeads before the
depletion protocol. A purity of more than 94% B220-IgM plasma cells was
achieved. Wild-type IgM⫹ B cells used in Western blot analysis and
adhesion assays were isolated from wild-type C57BL/6 spleens using
anti-IgM microbeads and LS⫹ selection columns (Miltenyi Biotec).
Fluorescence-activated cell sorting, DNA content
staining, and immunohistochemistry
Fluorescence-activated cell sorting (FACS) analysis was performed as
described elsewhere.12 For 3-color FACS analysis of E/P⫺/⫺ B-cell
compartments, various biotinylated monoclonal antibodies (mAbs) followed by a phycoerythrin (PE)–streptavidin second step were used in
conjunction with allophycocyanin (APC)–conjugated B220 and fluorescein
isothiocyanate (FITC)–conjugated anti-IgM. Data were collected using a
FACSort or FACSCalibur flow cytometer (Becton Dickinson, Mountain
View, CA). The following antibodies plus isotype controls were purchased
from Pharmingen (San Diego, CA): APC-RA3/6B2 (anti-CD45R/B220),
biotin-1D3 (anti-CD19), biotin-281.2 (anti-syndecan-1/CD138), biotin-90
(anti-CD38), biotin-KH74 (anti–major histocompatibility complex [MHC]
class II I-Ab), biotin-104 (anti-CD45.2), biotin-C71/16 (anti-CD18), biotinM17/4 (anti-CD11a), biotin-R1-2 (anti-CD49d), PE-MEL-14 (anti-CD62L/
L-selectin), and PE-IM7 (anti-CD44). FITC-conjugated and biotinylated
anti-IgM (b76), and biotinylated anti-CD43 (S7 and S11) were provided by
Dr Thomas Waldschmidt (University of Iowa, Iowa City). Anti–P-selectin
glycoprotein ligand 1 (PSGL-1) mAb 3C12 was provided by Immunex
(Seattle, WA). FITC-conjugated hyaluronic acid (HA) was provided by Dr
Mark Siegelman (University of Texas Southwestern Medical School,
Dallas). PE-conjugated streptavidin was purchased from Southern Biotechnology (Birmingham, AL) and APC-conjugated streptavidin was purchased
from Pharmingen.
For morphology analysis and immunohistochemistry, isolated plasma
cells were cytospun onto slides and fixed with ice-cold 95% ethanol/5%
glacial acetic acid. The fixed cells were then stained with either hematoxylin and eosin, or with mAb specific for Ig␬, Ig␭, IgA, or with polyclonal
anti-IgG or anti-IgM (Biosource, Camarillo, CA).
For cell cycle analysis, plasma cells were resuspended in 0.5 mL stain
solution, then passed through an 18-gauge needle and incubated in a shaker
for 20 minutes at 37°C. Then 0.5 mL of hypertonic solution was added, the
cells were again passed through an 18-gauge needle, wrapped in aluminum
foil, and stored at 4°C for 6 hours to overnight prior to FACS analysis.
Sodium dodecyl sulfate–polyacrylamide gel electrophoresis
and Western blot analysis
Sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE)
and Western blot analysis of plasma cell and IgM⫹ B-cell lysates were
performed as described.13 Cell lysates were made from equal numbers of
cells with the following high-salt RIPA lysis buffer: 50 mM Tris, pH 8, 150
mM NaCl, 1% Triton X-100, 1% deoxycholate (DOC), 0.1% SDS, 1 mM
In 96-well Unifilter plates (Whatman, Clifton, NJ), a mAb specific for
␬-light chain (Southern Biotechnology) was plated at 5 ␮g/mL overnight at
4°C in a humidified chamber. Prior to adding the cells, the plates were
washed 4 times with phosphate-buffered saline (PBS) and blocked for at
least 1 hour with Dulbecco modified Eagle medium (DMEM)/1% bovine
serum albumin (BSA) at room temperature. The cells were incubated on the
plates for 3 to 4 hours at 37°C. For purified plasma cells, 50 cells were
added per well, and 4000 spleen, bone marrow, or peripheral blood cells
were added per well. Each sample was analyzed in triplicate. Following 3
washes with PBS and 4 washes with PBS/Tween, 1:2000, 100 ␮L of the
appropriate biotinylated detecting antibody in PBS/Tween 1:2000/1% BSA
was added at 5 ␮g/mL and incubated overnight at 4°C in a humidified
chamber. The plates were then washed 4 times with PBS/Tween and
incubated with alkaline phosphatase conjugated-antibiotin antibody (Vector
Labs, Burlingame, CA) at a 1:1000 dilution in PBS/Tween/BSA for 2 hours
at room temperature. Following 4 washes with PBS, 200 ␮L developer (0.3
mg/mL nitroblue tetrazolium [NBT; Bio-Rad, Hercules, CA] and 0.15
mg/mL 5-bromo-4-chloro-3-inodolyl phosphate [BCIP; Sigma, St Louis,
MO] in Tris buffer) was added per well for approximately 15 minutes, and
the plates were washed 4 times with PBS, air dried, and counted using an
ELISPOT plate reader and software (Cellular Technologies, Cleveland, OH).
Low-shear L/VCAM-1 adhesion assay
L cells stably transfected with human VCAM-1 were plated in 35-mm
tissue culture plates at 150 000 cells/plate and allowed to grow to near
confluence. Then 0.70 ⫻ 106 to 1.0 ⫻ 106 E/P⫺/⫺ plasma cells or wild-type
IgM⫹ B cells were resuspended in 600 ␮L unsupplemented RPMI and
incubated with or without 50 nM phorbol myristate acetate (PMA) for 15
minutes at 37°C. After each L-cell plate was washed 3 times with RPMI, the
appropriate cells were added and incubated for 15 minutes on a continuously rocking platform at room temperature. Following incubation, each
plate was washed 5 times with RPMI and fixed with cold 0.74%
formaldehyde/RPMI solution. Where indicated, untransfected L cells were
used. For the blocking experiments, the plasma cells were incubated with 2
␮g anti-␣4 antibody (R1-2) in a volume of 100 ␮L for 10 minutes, following
treatment with 50 nM PMA or media alone. The volume was then increased
to 600 ␮L and the cells were immediately added to the L-cell plates and the
assay was continued as above. Cells bound per field were counted for 20
fields. The T-lymphoblastoid cell line, Jurkat, which binds well to VCAM-1,
was used as a positive control.
Parallel plate flow assay
Monolayers of Chinese hamster ovary (CHO) cells stably transfected with
either E-selectin or P-selectin were grown in 35-mm tissue culture plates
and served as the rolling substrate. Wild-type bone marrow cells, wild-type
IgM⫹ B cells, or E/P⫺/⫺ plasma cells were introduced into the flow chamber
(Glycotech, Rockville, MD) at a concentration of 0.5 ⫻ 106 cells/mL in
RPMI supplemented with 0.1% serum. Wild-type bone marrow cells (about
50% Gr-1⫹ neutrophils) roll well on both E-selectin and P-selectin and
served as the positive control. The shear stress in the flow chamber was
maintained constant at 1.5 dynes/cm2 using a syringe pump (Harvard
Apparatus, Holliston, MA), and images were obtained using a Nikon
Eclipse TE300 inverted microscope (Nikon, Melville, NY). Data analysis
was performed using Celltrak software developed by Compix (Cranberry
Township, PA), as previously described.14 Briefly, a rolling event is defined
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BLOOD, 15 APRIL 2002 䡠 VOLUME 99, NUMBER 8
IgG PLASMA CELL ADHESION PROFILE
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as a rolling cell that can be tracked between sequential images separated by
a 2-second time delay. The total number of rolling events was collected for
50 to 100 sequential images and the percentage of control events was
calculated. Data are represented as the mean percentage control of 3
experiments.
Glycosyltransferase reverse transcriptase–polymerase
chain reaction
Analysis of ␣1,3-fucosyltransferase-VII (FucT-VII), core 2 ␤1-6-Nglucosaminyltransferase (C2GlcNAcT-I), and dihydrofolate reductase
(DHFR) messenger RNA (mRNA) expression was performed using reverse
transcriptase–polymerase chain reaction (RT-PCR).15 Total cellular RNA
was isolated using Trizol (Life Technologies, Rockville, MD) from
wild-type bone marrow cells, wild-type IgM⫹ B cells, and E/P⫺/⫺ plasma
cells, and was used as a template in a 20-␮L RT reaction. RNA from equal
numbers of cells, ranging from 0.3 ⫻ 106 to 0.8 ⫻ 106 cells, was used in the
RT reactions. Wild-type bone marrow cells served as a positive control for
all genes, and DHFR served as an internal normalization control. Reactions
performed in the absence of RT were used as negative controls. A 50-␮L
PCR reaction was performed with cycling parameters 94°C/60°C/72°C for
30 seconds each with 32, 28, and 28 cycles for FucT-VII, C2GlcNAcT-I,
and DHFR, respectively, with primers as described.15 PCR products were
run out on 1.4% agarose gels, transferred to nitrocellulose, and
Southern blotted.
Statistical analysis
Differences between groups in the adhesion assays were analyzed using a
Student t test, with P ⬍ .05 being considered statistically significant.
Figure 1. Expression of lineage markers within the expanded E/Pⴚ/ⴚ lymph
node B cell compartment. (A) Non-B (CD5⫹/Mac-1⫹) cells were depleted from
E/P⫺/⫺ and control cervical lymph nodes as described in “Materials and methods.”
Examination of B220 and surface IgM expression by flow cytometry revealed 3
distinct B-cell populations present in E/P⫺/⫺ lymph nodes (indicated as I, II, and III),
but only the normal IgM⫹ B cells in wild-type lymph nodes. (B) Three-color flow
cytometry analysis was performed to further characterize the 3 E/P⫺/⫺ B-cell
populations. The histograms show expression of CD19, CD38, syndecan-1, MHC
class II, CD43 (S7), CD43 (S11), CD45, and an isotype control antibody on population
I (black line), population II (gray line), and population III (filled). A representative
experiment of at least 6 is shown. Fourteen total experiments examined syndecan-1
expression of population III cells resulting in an overall mean of 42.5% ⫾ 25.9%
positive. One representative experiment is shown.
Results
Cellular expansion and differentiation within the B-cell
compartment of E/Pⴚ/ⴚ cervical lymph nodes
The lymphadenopathy and the presence of morphologically defined
plasma cells observed in the cervical lymph nodes of E/P⫺/⫺ mice11
prompted us to investigate in detail the cellular composition of
these lymph nodes. Total cell numbers from the E/P⫺/⫺ cervical
lymph nodes were greatly increased compared to wild-type;
mean ⫾ SD was 54 ⫻ 106 ⫾ 19.9 ⫻ 106 (n ⫽ 86 mice, 22 isolations) for E/P⫺/⫺ mice, and ranged from 17.8 ⫻ 106 to 101.3 ⫻ 106
per mouse. For wild-type, the mean ⫾ SD per mouse was
5.3 ⫻ 106 ⫾ 3.2 ⫻ 106 (n ⫽ 36 mice, 7 isolations). Although the
expansion involved several cell types, including T cells and
macrophages, we focused our analysis on the B-cell compartment.
Depletion of non-B (CD5⫹/Mac-1⫹) cells revealed an expanded
B-cell compartment comprising 17.1% ⫾ 9.9% (n ⫽ 6) of total
cervical lymph node cells. Three main populations defined by
correlated B220 and surface IgM expression were evident (Figure
1A). Population I cells were B220hi and IgM⫹, corresponding to the
single population of normal B cells present in wild-type mice
(Figure 1A). As expected, these cells were CD19⫹, CD38⫹, MHC
class II⫹, syndecan-1/CD138⫺, and mostly negative for 2 CD43
epitopes, S7 and S11, with a subset of positive cells for each
(Figure 1B). Population II cells, not detectable in wild-type mice,
were B220hi and IgM⫺ (Figure 1A). These cells were also
uniformly CD19⫹, MHC class II⫹, S7⫺, and syndecan-1⫺, but
contained a distinct subset of CD38⫺ cells, and showed an increase
in S11 expression relative to the IgM⫹ cells (Figure 1B). In the
mouse, CD38 expression is lost in germinal centers, but is regained
in the transition to memory cells.16 Furthermore, the B220hi/IgM⫺
cells were surface IgG⫹ (data not shown). Taken together, this
phenotype of the B220hi/IgM⫺ population is consistent with a
combination of class-switched IgG memory cells and germinal
center cells.
Purified B220ⴚ/surface IgMⴚ cells are IgG plasma cells
Population III cells were negative for both B220 and surface IgM
(Figure 1A). In contrast to the 2 B220hi populations, these cells
were CD38⫺, MHC class II⫺, and CD19 expression was retained
by only a small subset (Figure 1B). In addition, these cells were
both S7hi and S11hi, and a subset (42.5% ⫾ 25.9%, n ⫽ 14)
expressed syndecan-1. Despite absent B220, the cells in population
III expressed equal levels of CD45 as the 2 B220hi populations
(Figure 1B). These cells also exhibited increased forward light
scatter (data not shown). This phenotype suggested that these cells
represent the plasma cells previously observed in E/P⫺/⫺ cervical
lymph nodes.11
We isolated the B220⫺/IgM⫺ population III cells to confirm that
this population consisted of plasma cells. These purified B220⫺/
IgM⫺ cells displayed classical plasma cell morphology, with
abundant dark staining cytoplasm and eccentrically placed nuclei
(Figure 2A). Furthermore, they were virtually all cytoplasmic (c)
Ig⫹, with about 97% cIgG⫹␬⫹ (Figure 2B), and only less than 4%
cIgA⫹, cIg␭⫹ or cIg␮⫹ cells detectable (Figure 2B and data not
shown). This profile is consistent with the previous observation of
increased IgG, but not IgM or IgA, levels in E/P⫺/⫺ serum,11 and
the approximately 20-fold higher frequency of ␬-expressing versus
␭-expressing B cells in normal mice.17,18 ELISPOT analysis
revealed that the plasma cells spontaneously secreted each of the
murine IgG subclasses, with the majority of cells secreting IgG1,
IgG2a, or IgG2b, and a small number of IgG3-secreting cells
(Figure 2C). Furthermore, cell cycle analysis using DNA content
staining with propidium iodide (PI) showed that approximately
97% of the cells were in the G0/G1 phase of the cell cycle (Figure
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2908
UNDERHILL et al
BLOOD, 15 APRIL 2002 䡠 VOLUME 99, NUMBER 8
Figure 2. B220ⴚ/IgMⴚ population III cells are IgGsecreting plasma cells. (A) B220⫺/IgM⫺ cells were
isolated as described in “Materials and methods,” cytocentrifuged onto slides, and stained with hematoxylin and
eosin. Original magnification ⫻ 40. (B) B220⫺/IgM⫺ cells
were purified, cytocentrifuged onto slides, fixed, stained
with FITC-conjugated anti-Ig␭, anit-Ig␬, anti-Ig␮, or antiIg␥ and examined by both phase and confocal microscopy. Original magnification ⫻ 40. (C) The number of
plasma cells secreting the various IgG isotypes was
determined by ELISPOT. The data are represented as the
mean (⫾ SEM) number of secreting cells per 50 cells for 4
independent plasma cell isolations. In each individual
experiment, each isotype was performed in triplicate. (D)
Purified plasma cells were fixed and stained with PI as
described in “Materials and methods,” and analyzed by
flow cytometry. A representative experiment of 3 is shown.
Cell lines examined in parallel showed significant fractions in the S and G2/M phases of the cell cycle (not
shown), whereas the plasma cells were nearly all G0/G1.
2D). Taken together, the surface phenotype, characteristic morphology, lack of cycling, and spontaneous secretion of antibody clearly
demonstrate that the B220⫺/IgM⫺ population present in the E/P⫺/⫺
cervical lymph nodes consists of IgG plasma cells.
blood from E/P⫺/⫺ mice were analyzed by ELISPOT. Plasma cells
secreting antibodies of all 4 murine IgG subclasses were detected at
increased levels compared with wild-type from each of these
E/P⫺/⫺ tissues (Figure 4A). Interestingly, the total frequency of
IgG-secreting plasma cells is highest and most elevated versus
IgG plasma cells express Blimp-1 but not BCL-6
We also examined the expression of 2 transcription factors
associated with different stages of B-cell differentiation, Blimp-1
and BCL-6, by Western blot analysis.19-22 As expected, the E/P⫺/⫺
plasma cells expressed Blimp-1, which is thought to be an
important transcription factor involved in plasma cell differentiation,19,20 whereas IgM⫹ B cells from wild-type spleens did not
(Figure 3). In contrast, neither the plasma cells nor the IgM⫹ B cells
expressed the transcriptional repressor BCL-6, which is expressed
principally in germinal center cells (Figure 3).21,23,24 The lack of
BCL-6 provides further evidence that the plasma cells have
completely exited the germinal center reaction.
IgG-secreting plasma cells are present in
multiple E/Pⴚ/ⴚ tissues
To quantify the extent of IgG plasma cell accumulation in other
E/P⫺/⫺ lymphoid tissues, bone marrow, spleen, and peripheral
Figure 3. Plasma cell expression of transcription factors. The expression of
Blimp-1 and BCL-6 by E/P⫺/⫺ plasma cells or wild-type IgM⫹ B cells was determined
by Western blot using rabbit polyclonal antiserums. The murine plasmacytoma cell
line, J558, served as the positive control (⫹) for Blimp-1 expression and BJAB cells
served as the positive control (⫹) for BCL-6 expression. A representative experiment
of 2 independent plasma cell and IgM⫹ B cell isolations is shown.
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BLOOD, 15 APRIL 2002 䡠 VOLUME 99, NUMBER 8
IgG PLASMA CELL ADHESION PROFILE
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of adhesive interactions, including rolling interactions under flow.28
A subset of purified plasma cells (24.8% ⫾ 16.4%, n ⫽ 8) stained
positively with HA-FITC (Figure 5B), similar to activated T cells,28
suggesting that these cells may be more capable of homing to bone
marrow or sites of inflammation than their HA-nonbinding counterparts. In contrast, no significant HA binding was detected in the
B-cell compartment before plasma cell purification (Figure 5B).
Acquisition of HA binding is therefore also plasma cell specific.
Plasma cells spontaneously bind to VCAM-1
Figure 4. Increased frequency of plasma cells in other E/Pⴚ/ⴚ tissues. ELISPOT
analysis was performed to quantify plasma cell numbers in E/P⫺/⫺ peripheral blood,
spleen, and bone marrow. (A) Four thousand leukocytes of each tissue type were
added per well and each dot represents the mean number of antibody-secreting cells
(ASCs) from triplicate wells for an individual mouse. For peripheral blood and spleen,
wild-type (WT) n ⫽ 6 and E/P n ⫽ 14, and for bone marrow, wild-type n ⫽ 9 and E/P
n ⫽ 19. The mean for each isotype is represented by a horizontal line. Note the scale
difference for peripheral blood. (B) The total number of IgG-secreting plasma cells for
wild-type versus E/P⫺/⫺ tissues is represented as the mean (⫾ SEM) ASCs per 4000
input leukocytes per mouse. Error bars are present for each group, although SEM is
too small to visualize for wild-type tissues and E/P⫺/⫺ bone marrow.
To investigate the functionality of the up-regulated ␣4 on the
plasma cells, we performed a low-shear adhesion assay using L
cells stably transfected with VCAM-1. Isolated plasma cells bound
about 5-fold better than IgM⫹ B cells (Figure 6A). Following
activation with PMA, plasma cell binding to the VCAM-1/L cells
increased by 70% to 80% and was still about 3.5-fold better than
IgM⫹ B cells (Figure 6A). Binding was specific because plasma
cell binding was reduced to background levels following the
introduction of an anti-␣4 blocking antibody or if untransfected L
cells were used (Figure 6B). Plasma cell binding to VCAM-1–
expressing cells was similar to the binding of the Jurkat T
lymphoblastoid cell line, with or without activation with PMA
(Figure 6C). These data demonstrate that ␣4 integrins up-regulated
by plasma cells are constitutively active and can mediate binding of
plasma cells to VCAM-1–expressing cells.
Selective rolling of plasma cells on E-selectin
wild-type in the peripheral blood compared with the spleen and
bone marrow (Figure 4B), suggesting an inability of these plasma
cells to exit the circulation due to the lack of the endothelial selectins.
We examined the interaction of plasma cells with E-selectin and
P-selectin under flow conditions in a parallel plate flow chamber at
IgG plasma cells up-regulate expression of multiple
leukocyte adhesion molecules
To investigate the adhesion characteristics of plasma cells, we first
examined the surface expression of leukocyte adhesion molecules
by the 3 B-cell subpopulations present in the cervical lymph nodes
of E/P⫺/⫺ mice. Three-color FACS analysis demonstrated that the
B220hi IgM⫹ population I and B220hi IgM⫺ population II cells had
equally low levels of leukocyte function-associated antigen (LFA-1;
CD11a/CD18) and ␣4 integrins (Figure 5A). Compared to the IgM⫹
cells, the B220hi IgM⫺ population had a larger subset of L-selectin
cells, consistent with previous activation, and a modest decrease of
PSGL-1 (Figure 5A). In contrast, the plasma cells showed a strong
up-regulation of ␣4 integrins, LFA-1, and PSGL-1, and failed to
express L-selectin (Figure 5A). Activation of B cells is therefore
not uniformly associated with up-regulation of leukocyte integrins,
in contrast to T cells. These results suggest that up-regulation of
both leukocyte integrins and PSGL-1 is characteristic of commitment to plasma cell differentiation, and not solely a consequence of
B-cell activation.
Cell surface expression of CD44 was increased on population II
cells, as expected, and on plasma cells (Figure 5B). CD44 is
involved in progenitor cell–stromal cell interactions in bone
marrow,25,26 as well as in homing of T cells to sites of inflammation.27 Increased expression of CD44 is characteristic of prior
activation for both B and T cells, but does not predict the
HA-binding properties of the cells. To examine the functionality of
the enhanced CD44 expression by the plasma cells, we analyzed
the binding of FITC-conjugated hyaluronic acid (HA-FITC) to the
plasma cells using flow cytometry.27,28 Previous work has demonstrated that binding of HA-FITC correlates well with the formation
Figure 5. Up-regulation of leukocyte adhesion molecules by plasma cells. (A)
Three-color flow cytometry analysis of the E/P⫺/⫺ cervical lymph node B-cell
populations (Figure 1) shows the relative expression of CD49d (␣4), CD18 and
CD11a (LFA-1), PSGL-1, L-selectin, and an isotype control antibody. The populations
are as in Figure 1. A representative experiment of 6 is shown. (B) On the left the
relative expression of CD44 on the 3 B-cell populations is shown. On the right a
subset of purified plasma cells bind HA-FITC. A representative experiment of 8 is
shown. HA-FITC staining is depicted on purified plasma cells (filled) versus the entire
B-cell compartment (CD5⫺, Mac-1⫺; negative control, gray line). For the experiment
shown, plasma cells constituted a small fraction of the entire B-cell compartment. An
HA-binding T cell line, BW5147, was used as a positive control (dotted line).
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BLOOD, 15 APRIL 2002 䡠 VOLUME 99, NUMBER 8
UNDERHILL et al
P-selectin nonbinding phenotype is distinct from that of activated T
cells, which bind well to both endothelial selectins.15,29
Plasma cells up-regulate FucT-VII but
down-regulate C2GlcNAcT-I
To determine the basis for this unique selectin-binding profile, we
examined the expression of glycosyltransferases involved in selectin ligand biosynthesis. Expression of FucT-VII is absolutely
required for formation of both E-selectin and P-selectin ligands,30
whereas C2GlcNAcT-I is essential for P-selectin binding,31,32 but is
not required for E-selectin binding under these conditions.33 We
therefore analyzed the mRNA levels of these enzymes in purified
plasma cells by RT-PCR. The plasma cells exhibited significantly
higher levels of FucT-VII mRNA relative to IgM⫹ B cells (Figure
8). In contrast, the plasma cells exhibited sharply decreased levels
of C2GlcNAcT-I mRNA (Figure 8), consistent with decreased
surface expression of B220, a C2GlcNAcT-I–dependent epitope.32
These results indicate that the selective interaction of plasma cells
with E-selectin under flow conditions is a function of the enhanced
levels of FucT-VII expression and that C2GlcNAcT-I is not
required for this interaction.
Figure 6. Enhanced binding of plasma cells to VCAM-1 through ␣4 integrins. (A)
Binding of plasma cells and IgM⫹ B cells to VCAM-1/L cells was analyzed using a low
shear rocking adhesion assay. Where indicated, cells were incubated with 50 nM
PMA for 15 minutes prior to the assay. (B) Plasma cell binding to untransfected L cells
(striped bars), or in the presence of 2 ␮g of the anti-␣4 blocking antibody R1-2 (empty
bars). Data are represented as the mean number of bound cells per field ⫾ SD for 20
fields of view. A representative experiment of 3 is shown. The asterisk indicates
statistically different (P ⬍ .05) from part A, the corresponding group (with or without
PMA) of plasma cells; or in part B, plasma cells binding to VCAM-1 in the absence of
blocking mAb. (C) The relative binding of plasma cells and Jurkat cells. The mean ⫾
SD of 2 independent experiments is shown.
a shear stress of 1.5 dynes/cm2. The isolated plasma cells rolled
well on E-selectin, with the number of interactions equal to
43.1% ⫾ 13.7% (n ⫽ 3) of bone marrow cells (about 50% neutrophils; Figure 7A). Bone marrow leukocytes represent a population
capable of significant rolling interactions on E-selectin and Pselectin.5 In contrast, very little plasma cell rolling was observed on
P-selectin (9.3% ⫾ 11.2% of control, n ⫽ 3; Figure 7B), despite
the elevated levels of PSGL-1 protein (Figure 5A). B cells did not
roll detectably on E-selectin and rolled only slightly on P-selectin
(5.3% ⫾ 8.4% of control, n ⫽ 5; Figure 7). This E-selectin binding/
Figure 7. Plasma cells interact selectively with E-selectin. Plasma cells and IgM⫹
B cells were analyzed for interactions with E-selectin (A) or P-selectin (B) transfected
CHO cells in a parallel plate flow chamber, as described in “Materials and methods.”
The data are represented as a mean percent control interactions ⫾ SD (n ⫽ 3), with
wild-type bone marrow cells serving as positive control. Asterisk indicates statistically
different (P ⬍ .05) from IgM⫹ B cells; NS, not statistically different.
Discussion
Tissue-specific leukocyte homing is mediated by the regulated
expression of adhesion molecules as well as differential responsiveness to chemotactic signals and has been studied extensively for
multiple cell types. For example, Th1 and Th2 effector cells display
distinct homing patterns resulting in part from differential expression of glycosyltransferases and consequently selectin ligands.29,34
However, less is known about the homing of B-cell effector cell
types. Specifically, the mechanisms that control plasma cell
migration and anatomic localization remain largely unknown. To
examine plasma cell adhesion and homing molecules we isolated
IgG plasma cells from the cervical lymph nodes of E/P⫺/⫺ mice.
The analysis of the E/P⫺/⫺ cervical lymph nodes revealed a
B-cell compartment comprised of cells at multiple stages of
differentiation (Figure 1). In addition to the unactivated IgM⫹ B
cells (population I) corresponding to the single population in
wild-type mice, 2 additional major populations were identified in
Figure 8. Plasma cells up-regulate FucT-VII but down-regulate C2GlcNAcT-I.
Expression of FucT-VII and C2GlcNAcT-I mRNA by E/P⫺/⫺ plasma cells and
wild-type IgM⫹ B cells was determined by RT-PCR. Wild-type bone marrow was used
as a positive control for all 3 genes, and DHFR served as a normalization control.
Presence or absence of RT in the RT-PCR reactions is indicated. A representative
experiment of 3 is shown.
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BLOOD, 15 APRIL 2002 䡠 VOLUME 99, NUMBER 8
the E/P⫺/⫺ mice. Population II cells were B220hi and IgM⫺,
expressed CD19, MHC class II, surface IgG, and consisted of
CD38⫹ and CD38⫺ subsets as well as L-selectin⫹ and L-selectin⫺
subsets (Figures 1 and 5A, and data not shown). This phenotype is
consistent with a combination of germinal center cells and postgerminal center IgG memory cells. Population III consisted of plasma
cells, which were negative for B220 and surface IgM, as well as
CD38 and MHC class II (Figure 1). These plasma cells were
essentially all cIgG⫹, spontaneously secreted antibody, and were
similar morphologically (Figure 2). The presence of B cells at
distinct stages of differentiation ranging from IgM⫹ unactivated
cells to IgG-secreting plasma cells within the E/P⫺/⫺ lymph nodes
provides a unique view into antigen-driven B-cell differentiation.
The E/P⫺/⫺ plasma cells exhibited heterogeneity with respect to
a few markers. For instance, several subclasses of IgG antibodies
were represented within the plasma cell population (Figure 2C).
The diversity of IgG isotypes is expected due to the bacterial
infections, which are persistent in E/P⫺/⫺ mice.7,11 Additionally,
only a subset of these IgG plasma cells expressed syndecan-1
(42.5% ⫾ 25.9%, n ⫽ 14; Figure 1B). Syndecan-1 has been used
previously as a marker for IgM-secreting plasma cells in vitro and
in vivo, and for IgG1-secreting plasma cells in the spleen and bone
marrow following immunization with the hapten (4-hydroxy-3nitrophenyl) acetyl (NP).35-37 Our results suggest that syndecan-1
may not be expressed on all plasma cells, or on plasma cells at all
stages of differentiation. Furthermore, a small subset of plasma
cells retained CD19 expression. The incomplete loss of CD19
expression has been observed previously for antibody-secreting
cells following immunization with NP.37 Taken together, the
retention of CD19 expression by a fraction of cells and the
heterogeneous expression of syndecan-1 imply the existence of
multiple distinct stages of plasma cell differentiation. The plasma
cells were also heterogeneous for binding of HA (Figure 5B). It is
therefore attractive to speculate that expression of syndecan-1 or
the ability to bind HA may be a marker for long-lived plasma cells
destined to home to and reside in bone marrow.
High numbers of plasma cells have also been reported in the
lymph nodes of mice deficient in CD18 or CXCR2. Similar to the
E/P⫺/⫺ mice, CD18⫺/⫺ and CXCR2⫺/⫺ mice display highly elevated leukocyte counts and severe defects in neutrophil recruitment to sites of inflammation, in addition to an accumulation of
plasma cells in their cervical lymph nodes.38,39 Both the E/P⫺/⫺ and
CD18⫺/⫺ mice also suffer from progressive skin lesions that are
secondary to unresolved dermal infections with endogenous bacteria. Consequently, mice with targeted mutations in different genes,
each of which results in severely compromised neutrophil traffic,
result in strikingly similar phenotypes. This observation supports
the idea that the large expansion and differentiation of the B-cell
compartment, as well as other lymph node populations, in these
animals results from persistent bacterial infections that cannot be
effectively eliminated, due to severely impaired recruitment of
neutrophils. Thus, persistently high levels of antigen and, presumably, various bacterial products, continuously drives an intense
immune response.
In addition to the presence of plasma cells in the cervical lymph
nodes, increased IgG-secreting plasma cells were detected in
spleen, bone marrow, and peripheral blood of E/P⫺/⫺ mice (Figure
4). In wild-type mice, the highest frequency of IgG plasma cells
was in the bone marrow, with extremely low levels detected in
wild-type peripheral blood, as expected. In contrast, peripheral
blood from E/P⫺/⫺ mice contained a higher frequency of IgG
plasma cells than either E/P⫺/⫺ spleen or bone marrow. These data
IgG PLASMA CELL ADHESION PROFILE
2911
illustrate that enhanced plasma cell differentiation or accumulation
in E/P⫺/⫺ mice is not restricted to the cervical lymph nodes.
Furthermore, the substantial increase in peripheral blood plasma
cells in E/P⫺/⫺ mice serves as preliminary evidence for a possible
role of E-selectin or P-selectin in plasma cell migration.
Our results demonstrate that IgG plasma cells display a unique
array of adhesion molecules involved in leukocyte traffic. Specifically, these plasma cells showed a greatly increased expression of
␣4 integrins, LFA-1, CD44, and PSGL-1 (Figure 5). For the
leukocyte integrins and PSGL-1, these were plasma cell–specific
changes not specifically associated with activation, because they
were not present on B220hi/IgM⫺/IgG⫹ B cells. Furthermore, the
␣4 integrins on plasma cells were shown to mediate substantial
binding of plasma cells to VCAM-1 (Figure 6), and a subset of
plasma cells bound HA (Figure 5B), indicating that these molecules
were active and functional. Up-regulation of leukocyte integrins
and CD44 is also characteristic of activated/memory T cells, but
up-regulation of PSGL-1 has not been previously observed on
normal activated lymphocytes.
Of equal importance, the purified plasma cells interacted
selectively with E-selectin but not P-selectin under flow conditions,
despite the higher levels of PSGL-1 protein (Figures 5 and 7). This
is in sharp contrast to activated T cells, particularly Th1 cells,
which exhibit high levels of rolling on both endothelial selectins.15,29,34 Plasma cells also showed an up-regulation of FucT-VII
mRNA (Figure 8), an enzyme required for E-selectin and P-selectin
ligand biosynthesis,30 but this increased expression of FucT-VII
mRNA was accompanied by a sharp decrease in C2GlcNAcT-I
mRNA levels (Figure 8). Recent work revealed an absence of
rolling on P-selectin but unimpaired rolling on E-selectin of
C2GlcNAcT-I⫺/⫺ leukocytes under the in vitro conditions used
here,33 demonstrating that C2GlcNAcT-I is selectively required for
functional P-selectin ligands. The preferential up-regulation of
FucT-VII accompanied by a down-regulation of C2GlcNAcT-I by
the plasma cells also contrasts with the up-regulation of both of
these enzymes in activated T cells, particularly Th1 cells.29 The
down-regulation of an enzyme selectively required for binding to
P-selectin but not E-selectin represents a novel mechanism for
control of lymphocyte traffic, and these plasma cells represent the
first such example.
E-selectin, P-selectin, and VCAM-1 are important in progenitor
cell rolling in bone marrow microvessels and recruitment of HPCs
to bone marrow following irradiation and transplantation.8,9 Both
human and murine bone marrow endothelial cells constitutively
express VCAM-1,6,40,41 human bone marrow endothelial cells
express E-selectin,6 and E-selectin has been detected in murine
bone marrow using RT-PCR.7 In vitro rolling experiments performed here revealed that purified plasma cells roll well on
E-selectin, but not P-selectin (Figure 7), and expressed upregulated ␣4 integrins that supported binding to VCAM-1 (Figures
5 and 6). Taken together, this suggests that the interaction of plasma
cells with E-selectin and VCAM-1 are likely to be important in the
bone marrow localization of these cells. In fact, the selective
interaction of the plasma cells with E-selectin, and not P-selectin,
may represent a mechanism for preventing the migration of plasma
cells to acute inflammatory sites, thereby directing them to the bone
marrow. Intact ␣4/VCAM-1 interactions, still present in E/P⫺/⫺
mice, as well as interactions mediated by other adhesion molecules,
such as CD44, most likely underlie the increased frequency of IgG
plasma cells in the bone marrow of E/P⫺/⫺ mice in the absence of
E-selectin (Figure 4). In vivo homing experiments using IgG
From www.bloodjournal.org by guest on June 17, 2017. For personal use only.
2912
BLOOD, 15 APRIL 2002 䡠 VOLUME 99, NUMBER 8
UNDERHILL et al
plasma cells purified from E/P⫺/⫺ mice will provide more information regarding the cooperative effects of these multiple adhesion
molecules in plasma cell bone marrow localization.
In conclusion, we have demonstrated that IgG plasma cells
display a unique constellation of leukocyte adhesion molecules.
Within the B-cell compartment, an adhesion phenotype specific to
plasma cell differentiation was observed, including up-regulation
of integrin expression, integrin activation, HA binding, and rolling
interactions with E-selectin. IgG plasma cells additionally exhibited a pattern of glycosyltransferases consistent with the preferential selectin mediated rolling capabilities observed for these cells.
Taken together, this plasma cell–specific spectrum of adhesion
molecules described here likely underlies the distinct homing and
anatomic localization of antibody-secreting plasma cells. The
further analysis of the IgG plasma cells, and in particular plasma
cell subsets, generated in E/P⫺/⫺ mice will offer further insight into
plasma cell differentiation mechanisms and possible markers for
bone marrow homing or long-lived plasma cells.
Acknowledgments
We thank Dr Tom Waldschmidt, University of Iowa, Iowa City, for
FACS reagents and helpful discussions, and Dr Mark Davis,
Stanford University, for Blimp-1 antiserum. We also thank Dr
Mark Siegelman, University of Texas Southwestern Medical
Center, Dallas, for FITC-HA.
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2002 99: 2905-2912
doi:10.1182/blood.V99.8.2905
IgG plasma cells display a unique spectrum of leukocyte adhesion and homing
molecules
Gregory H. Underhill, Heather A. Minges-Wols, Jamie L. Fornek, Pamela L. Witte and Geoffrey S. Kansas
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