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IMMUNOBIOLOGY
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.
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/P 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 constitutively 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 E-selectin and P-selectin double-deficient (E/P Mice
Cell isolation
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), biotin-M17/4 (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 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 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 In 96-well Unifilter plates (Whatman, Clifton, NJ), a mAb specific for -light chain (Southern Biotechnology) was plated at 5 µg/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 µL of the appropriate biotinylated
detecting antibody in PBS/Tween 1:2000/1% BSA was added at 5 µg/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 µL 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 µL 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 µg
anti- 4 antibody (R1-2) in a volume of 100 µL for 10 minutes, following treatment with 50 nM PMA or media alone. The volume
was then increased to 600 µL 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 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 of1,3-fucosyltransferase-VII (FucT-VII), core 2  1-6-N-glucosaminyltransferase (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-µL 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-µL 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.
Cellular expansion and differentiation within the B-cell
compartment 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 B220 , 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
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/P / lymphoid tissues, bone marrow, spleen, and
peripheral 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
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.
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 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 To investigate the functionality of the up-regulated4 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 We examined the interaction of plasma cells with E-selectin and P-selectin under flow conditions in a parallel plate flow chamber at 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 P-selectin.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/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.
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 The analysis of the E/P The E/P 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 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 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 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 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 up-regulated
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
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 FIT |
Top
Abstract
Introduction
, IgA, or
with polyclonal anti-IgG or anti-IgM (Biosource, Camarillo, CA).
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.
