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Sir William Dunn School of Pathology, University of Oxford, Oxford, United Kingdom
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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T cells are thought to monitor the surface of DC for antigenic MHC-peptide complexes via undefined non-Ag-specific adhesion interactions (6), represented in vitro by Ag-independent DC/T cell clustering. We have found that DC can make similar Ag-independent interactions with resting B cells, independently of T cells, and suggest that this interaction may have important functions in the induction of Ab synthesis.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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PVG (RT1c) and DA (RT1a) specific pathogen-free rats were bred at the Medical Research Council (MRC) Cellular Immunology Unit, Sir William Dunn School of Pathology, Oxford. U.K. Surgical procedures (mesenteric lymphadenectomy (MLNX) and thoracic duct cannulation) were as described previously (7). Thoracic duct lymph (TDL) cells were collected from cannulated MLNX rats over 3 days.
Reagents
Culture medium was RPMI 1640 supplemented with 2 mM
L-glutamine, 1 mM sodium pyruvate, 10% FCS,
antibiotics, and 25 µM mercaptoethanol. To generate DC in bone marrow
cultures, 5% FCS was used and mercaptoethanol was not added. Sodium
metrizamide and NycoPrep were purchased from NycoMed (Oslo, Norway).
Isopaque-Ficoll was prepared from Ficoll (Pharmacia, Sweden) and sodium
metrizoate (Nyegaard, Oslo, Norway). LPS (Salmonella
typhimurium) was from Sigma Chemical Co. (St. Louis, MO).
Collagenase A and DNase I were from Boehringer Mannheim (Mannheim,
Germany). Percoll was from Pharmacia. PMA, D-sphingosine,
genistein, cytochalasin B, cycloheximide, EDTA, EGTA, sodium azide, and
glutaraldehyde were purchased from Sigma, nocodazole from Calbiochem.
3,3'-Diaminobenzidine tetrahydrochloride (DAB) was obtained from
Polyscience (Warrington, PA). Recombinant murine GM-CSF was a gift from
British Biotechnology (Oxford, U.K.) (spec. act., 0.7–2 x
107 U/mg). Murine rTNF-
, IFN-
(spec. act.,
107 U/mg), and human ultrapurified TGFß were a kind gift
from Prof. S. Gordon (Sir William Dunn School of Pathology). Rat rIL-4
as tissue culture supernatant from an IL-4-transfected CHO cell line
(8) was kindly provided by Dr. D. Mason (Sir William Dunn School of
Pathology).
Antibodies
Most monoclonal mouse anti-rat Abs were kind gifts from the
MRC Cellular Immunology Unit, Sir William Dunn School of Pathology.
OX62 mAb (mouse anti-rat DC integrin) was a kind gift from Dr. M.
Brenan (9). WT.1 (anti-CD11a), WT.3 (anti-CD18), 1A.29
(anti-CD54), and HRL3 (anti-CD62L) anti-rat Abs were kindly
provided by Prof. M. Miyasaka (Tokyo, Japan). HP2/1 (anti-CD49d) Ab
(mouse anti-human, cross-reacts with rat) was a gift from Dr. A.
Ager (National Institute of Medical Research, London, U.K.). MARD3 and
OX77 anti-rat IgD Ab were gifts from by Dr. S. Hunt (Sir William
Dunn School of Pathology). MARA-1 Ab (anti-rat IgA) was from
Serotec (Kidlington, U.K.). Murine CTLA4-IgG2a fusion protein that
cross-reacts with rat B7 was a gift from Dr. P. Linsley (Bristol-Myers
Squibb, Seattle, WA). Rabbit anti-human CD22 Ab (cross-reacts with
rat) was a gift from Dr. P. Crocker (Institute of Molecular Medicine,
Oxford, U.K.). Peroxidase-conjugated rabbit anti-mouse Ig Ab (RAM)
was from Dako (Glostrup, Denmark). Goat anti-mouse Ig Ab (GAM)
absorbed with rat serum was from Sigma. FITC-conjugated OX12
(anti-
chain) and OX6 (anti-MHC class II) Abs and
phycoerythrin (PE)-conjugated F(ab')2
fragments of RAM were purchased from Serotec.
In vivo endotoxin administration
Normal or MLNX-cannulated rats were injected with 50 µg LPS in 1 ml PBS via a tail vein. TDL samples, spleens, or lymph nodes were collected at different time points; DC from TDL of MLNX-cannulated rats (XTDL) and B cells from the spleen and lymph nodes were examined for their ability to form clusters.
DC enrichment
Lymph-borne (veiled) DC (L-DC) were prepared from XTDL by density separation over 14.5% metrizamide solution or NycoPrep (7). DC from mesenteric lymph nodes (MLN), spleen, and PP were extracted as previously described (10). DC from lamina propria (LP) were isolated as described by Lawson et al. (11) with modifications. Briefly, fragments of PP-free intestine were inverted and pulled over glass rods. This was followed by incubation and vigorous vibration in 25 mM EDTA solution at 4°C. This was followed by digestion with collagenase-DNase for 60 min at 37°C (incubation and vibration) and another 30-min cycle of EDTA treatment at 37°C. DC were isolated from the cells released by enzymatic and the last EDTA treatments by metrizamide separation. Purity of DC preparations was evaluated on the basis of cell morphology in fresh samples and by the staining with OX62 and OX6 mAbs. The contaminating cells were mainly B lymphoblasts.
Lymphocyte separation
B cells and T cells were enriched by rosette depletion of T cells or B cells, respectively (10). mAbs W3/13 (CD43), OX8 (CD8), W3/25 (CD4), and OX52 (pan-T) were used for T cell depletion, OX6 (MHC class II) and OX12 (Ig L chain) for B cell depletion. SRBC coupled to GAM Ab were used for rosetting. TDL cells were obtained from the first overnight collection of lymph from cannulated rats. Spleen and lymph node cells were prepared by teasing organs into single-cell suspensions. Peripheral blood cells were prepared from blood obtained by cardiac puncture and separated over Isopaque-Ficoll. Bone marrow cells were obtained by flushing the marrow from the femur with washing medium.
To study the effect of activation on B cell clustering, a discontinuous Percoll gradient was used to separate small and large B lymphocytes. B cells were layered above the lightest Percoll fraction and the gradient consisting of four Percoll fractions (30, 45, 55, and 65%) and one cell fraction (2 ml each) was centrifuged for 20 min at 2000 rpm at room temperature. Lymphocytes localizing in the interface between 55 and 65% Percoll fractions were considered resting B cells.
Cluster formation assay
B cells and DC were cocultured for 2 to 3 h in Teflon pots (Savilex; Techmate, Milton Keynes, U.K.) in 0.5 ml of washing medium on a slowly rotating wheel at 37°C. The total cell number was 5 x 105. If not otherwise indicated, the B cell:DC ratio was 10:1. After culturing, cytospin (CSP) preparations were made, and cluster formation was assessed by direct counting of 100 to 200 free and clustered DC on CSPs stained using immunoperoxidase. The percentage of clusters was calculated as the ratio of clustered DC to the total number of counted DC on the CSP. In some experiments, the numbers of B cells adherent to 100 DC were also counted. These counts gave very similar results to those in which numbers of clustered DC were counted. To show that the clusters were not artifacts of the cytospin procedure, CSPs of DC and B cells were made immediately after mixing. These showed that only occasionally was a DC in contact with more than two B cells, and we thus defined clusters as one or more DC in contact with four or more B cells. If two or more DC were present in a cluster, individual DC were only counted as clustered if each one was in contact with at least four B cells.
Immunocytochemistry
A standard immunoperoxidase technique was used. Briefly, CSPs were fixed in absolute ethanol for 10 min and incubated with mAb tissue culture supernatants for 30 to 60 min at 4°C. After extensive washes, CSPs were incubated with the second-layer Ab, RAM-peroxidase, for 30 to 60 min at 4°C. DAB solution with H2O2 was used as a substrate. Slides were lightly counterstained with Erlich’s hematoxylin, dehydrated, and mounted in DPX (BDH, Poole, U.K.).
Flow cytometry
Lymphocytes and DC were double-labeled and analyzed on a FACScan (Becton Dickinson, Mountain View, CA). Briefly, cells were labeled with a primary Ab (tissue culture supernatant (TCS) or purified IgG at 0.25–0.5 µg per 106 cells), followed by incubation with F(ab')2 fragments of PE-conjugated RAM-IgG. MRC OX21 (anti-human factor I) and W6/32 (anti-HLA) mAbs served as isotype matching negative controls (IgG1 and IgG2a, respectively). F(ab')2 RAM-PE Ab was adsorbed with rat serum proteins and did not cross-react with rat B cells. To prevent the binding of a second-color mouse anti-rat mAb, cells were incubated with a saturating amount of mouse IgG (10 µg/ml final concentration). B cells were positively gated using FITC-conjugated MRC OX12 as a second-color mAb. DC were identified as the brightest population of cells stained with FITC-conjugated MRC OX6. Labeling with MRC OX33 mAb (B cell-restricted isotype of CD45) or MRC OX62 mAb (DC-specific integrin) confirmed that the majority of MRC OX12+ and MRC OX62+ cells were B cells and DC, respectively.
DC culture
Density gradient-purified DC were cultured for different periods of time in complete RPMI 1640 supplemented with 10% FCS at 1.5 to 2 x 105 DC in 0.2 ml in round-bottom 96-well plates (Falcon; Becton Dickinson, Lincoln Park, NJ) at 37°C. After culture, cells were collected and survival was estimated by a direct count of recovered DC before washing. DC were washed once and used in the cluster formation assay or for phenotype analysis.
Cell fixation
B cells or DC were washed gently in PBS and fixed with 0.125% glutaraldehyde (final concentration) for 10 min at room temperature. Cells were washed twice with PBS and incubated with PBS supplemented with 10% FCS for 10 min. After a single washing, cells were used in cluster formation assays.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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CSPs made after incubation of freshly isolated L-DC with
lymphocytes from spleen or TDL showed that some DC had formed clusters
with lymphocytes. The adherent lymphocytes were >95% B cells (Fig. 1
). To quantitate clustering, DC were
counted as clustered if 4 or more B cells were adherent. Under the
conditions used for cluster formation, most clusters were small, with 1
to 3 DC and 5 to 15 B cells. If more than 1 DC was present in a
cluster, a DC was only counted as clustered if 4 or more B cells could
be seen attached to it. To assess clustering in a way that would avoid
the possibility of skewing due to changes in DC/DC clustering, in some
experiments the number of B cells adherent to 100 DC was counted. The
two methods gave very similar results, and in no case did the
conclusions drawn from an experiment differ.
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). Clusters started to form within 15 min and peaked at 1 h.
Clusters, once formed, remained stable for some hours, but many had
dissociated by 24 h and none remained after 48 h. Clustering
is not MHC dependent, as DC clustered equally well with B cells from
syngeneic or allogeneic rats. Cross-linking of B cell surface Ig by Abs
to µ or chains or short term (4–6 h) stimulation with LPS did
not have any immediate effect on the ability of B cells to cluster DC,
suggesting an Ag-independent mechanism. B blasts in TDL do not cluster DC
To determine directly whether activation abolishes clustering, B
cells were activated in vitro by culture with LPS for 72 h. Small,
high density B cells remain able to cluster DC, whereas B cells from
the low density fractions form very few clusters (Fig. 2a), and those clusters
that do form involve only contaminant small B cells.
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b). By 96 h, splenic and lymph node B cells had
recovered their ability to cluster DC. The effect is not due to loss of
B cells from the spleen, as total B cell numbers recovered from the
spleen were similar in treated and untreated animals (data not shown).
The effect is indirect, as B cells cultured with LPS for 8 h did
not lose the ability to cluster DC (data not shown). The effect does
not result from the down-regulation of LFA-1 expression, as levels of
LFA-1 on splenic B cells are slightly increased (not shown),
suggesting involvement of other surface molecules. DC/B cell clusters form in vivo
Occasional clusters of lymphocytes and DC are present in fresh
lymph obtained directly from the cannula in MLNX rats (Fig. 1C). CSPs of such clusters show that most adherent
lymphocytes are B cells (not shown). When lymph was collected from
animals that had protein Ag injected into the small intestine at
laparotomy, there was a marked increase in the numbers of DC/B cell
clusters seen in fresh lymph by 24 h (not shown). The injection of
PBS had a similar but lesser effect.
Clustering is LFA-1 dependent
DC and B cells were cultured in the presence of Abs against
functional epitopes of surface molecules (Fig. 3). Of those available for the rat, only
mAbs against the - or ß-chains of LFA-1 (WT.1 and WT.3) inhibited
clustering significantly. The counter-receptor for LFA-1 in this
interaction is not ICAM-1, as a blocking anti-ICAM-1 mAb (1A.29)
did not reduce clustering (Fig. 3a). Depletion of
either LFA-1+ B cells or LFA-1+ DC decreased
clustering. When both LFA-1+ B cells and LFA-1+
DC were eliminated, only occasional clusters were seen (Fig. 3b). Thus, B cell/DC binding is at least partially
mediated by LFA-1. Clustering is cation dependent, as coculture of B
cells and DC in the presence of EDTA or EGTA inhibited cluster
formation. Reconstitution of the media with Ca2+ completely
restored cluster formation, whereas addition of Mg2+ had
little effect (Fig. 3c).
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DC/B cell clustering is not a passive interaction (Fig. 4). Thus sodium azide, fixation of DC or
B cells, or incubation at 4°C strongly inhibited cluster formation,
showing a need for metabolic activity. Treatment of cells with
cytochalasin B or nocodazole also inhibited clustering, demonstrating
cytoskeletal involvement (Fig. 4a).
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b). These data
demonstrate a requirement for PKC activation and further suggest the
involvement of integrins. Ability of B cells to cluster is up-regulated in lymph nodes and spleen
Most small B cells belong to a common recirculating pool but B
cells isolated from different tissues show consistent differences in
clustering; spleen and lymph node cells forming more clusters than
lymph or blood cells, with bone marrow B cells not clustering
significantly (Fig. 5a). The splenic B
cells that clustered were sIgM+ and mostly
sIgD+ (not shown), showing that they belonged to the
recirculating pool. Marginal zone B cells are mainly surface
IgD- and do not recirculate (12), but purified surface
IgD- splenic B cells also formed clusters with DC (data
not shown). The differences in clustering do not relate to levels of
LFA-1 expression, as flow cytometry showed only very small differences
between splenic and lymph B cells (data not shown).
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The ability of DC to cluster B cells is also variable and may
relate to their maturity (Fig. 5b). DC from spleen
and MLN were the most potent, L-DC were intermediate, and DC from PP
and LP had a relatively low potency (Fig. 5b). Not
all DC in a population are able to cluster B cells. Thus, when L-DC
were cultured with increasing numbers of B cells, the percentage of DC
forming clusters reached a plateau at 30 to 50%. No phenotypic
differences were detected between DC that did and did not form
clusters.
To determine whether the ability of DC to form clusters is a stable
characteristic, we attempted to form clusters with cultured DC. DC
extracted from all sources showed a marked decrease in adhesion to B
cells after 16 to 24 h of culture (Fig. 5b).
Short term culture (4 h) did not have any effect (data not shown). The
effects of culture cannot be explained by down-regulation of adhesion
molecules in culture, as L-DC cultured for 16 to 24 h showed no
significant difference in expression of LFA-1, ICAM-1, and OX62 (data
not shown).
DC were cultured in the presence of recombinant cytokines (murine
GM-CSF and TNF-, human TGFß, murine IFN-, and rat rIL-4).
Although GM-CSF and IL-4 increase DC survival in culture (13), (N.
Kushnir and G. G. MacPherson, unpublished observations), no cytokines,
either alone or in combination with GM-CSF, reversed the decrease in
the ability of cultured DC to form clusters (data not shown).
Cross-linking of Thy-1 or MHC class II with mAbs stimulates cluster formation
To further investigate the mechanisms regulating B cell/DC cluster
formation, we tested the effects of mAbs to cell surface molecules.
mAbs to MHC class II and Thy-1 caused a dramatic stimulation of B
cell/DC clustering (Fig. 6). This effect
is not a result of Ab-mediated passive cross-linking of cells, because
mAbs against MHC class I, CD45, and CD44, also expressed at high levels
on both cell types, do not increase cluster formation (Fig. 7a). In addition,
removal of B cells expressing Thy-1 did not decrease the ability of
anti-Thy-1 mAb to stimulate clustering (Fig. 7c).
mAb-induced aggregation is not mediated via FcR, since
F(ab')2 fragments of OX6 mAb had the same effect as whole
mAb (Fig. 7a).
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b). Treatment with sphingosine also blocked
mAb-induced clustering, suggesting a role for PKC. However, the
molecular basis of stimulated B cell/DC aggregation is not known;
it does not appear to be associated with LFA-1 or other
integrins, as neither anti-LFA-1 mAb nor EDTA had any inhibitory
effect (Fig. 7b).
To determine which cells were stimulated by anti-MHC class II mAb,
DC or B cells were separately incubated with OX6 mAb at 4°C followed
by washing and coculture with their untreated counterparts. Treatment
of either B cells or DC stimulated aggregation to the same extent as
when OX6 was present in the culture (data not shown). As Thy-1
expression varies on L-DC, and as a small proportion of B cells in TDL
express Thy-1, we could examine the role of Thy-1 by selective
depletion of Thy-1+ DC or B cells. Thy-1+ DC or
B cell subsets were depleted, and the Thy-1- cells were
cocultured with their unseparated counterparts in the presence of
anti-Thy-1 mAb. Removal of Thy-1+ TDL B cells did not
prevent anti-Thy1-induced adhesion, whereas in the absence of
Thy-1+ DC, no aggregation was observed (Fig. 7c). This experiment eliminates the possibility that
the increased clustering is due to passive agglutination of DC and B
cells. Taken together, these data show that cross-linking of MHC class
II or Thy-1 triggers signal transduction that results in potent
heterotypic aggregation of B cells and DC via an unknown adhesion
pathway.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Molecular basis of clustering
Adhesion of B cell to DC is not a passive process but depends on metabolic and cytoskeletal activity, and on PKC but not protein tyrosine kinase activation, showing that intracellular signaling is essential for clustering. Clustering in steady state conditions is LFA-1 dependent, being inhibited by anti-LFA-1 mAbs and depletion of LFA-1+ B cells and/or DC. The counter-receptor for LFA-1 is unknown, as a blocking anti-ICAM-1 mAb did not affect clustering. ICAM-3 is the major ICAM on human DC involved in the stimulation of resting T cells (16), but as neither polyclonal nor monoclonal anti-human ICAM-3 Abs react with rat leukocytes (N. Kushnir and G. G. MacPherson, unpublished observations), we cannot confirm ICAM-3 as the ligand for LFA-1.
The expression of LFA-1 by B cells is not sufficient to mediate adhesion to DC. Thus, B cells activated in vivo by LPS express identical (lymph node) or higher (spleen) levels of LFA-1, but clustering is reduced by >80%. Resting T cells express higher levels of LFA-1 than B cells but do not adhere to DC as efficiently. The increased clustering stimulated by cross-linking MHC class II or Thy-1 was not inhibited by anti-LFA-1 Abs and was not cation dependent. Thus, LFA-1 is not the only molecule involved in clustering. The identity of the other molecules involved in DC/B cell adhesion remains to be elucidated. If DC/B cell adhesion employs mechanisms similar to those involved in leukocyte-endothelium adhesion, we would predict the involvement of selectins, chemokines, and their receptors. L-selectin is not involved, as a blocking Ab did not interfere with clustering.
Regulation of clustering
Only small, resting B cells cluster DC, but most of these cells are part of a common recirculating pool, containing cells at different stages of maturity and at different points in their migratory pathways. Immature (bone marrow) B cells do not form clusters, and B cells from secondary lymphoid organs (spleen, lymph nodes, PP) are much more active than those from the lymph or blood. In addition, sessile, IgD- B cells from the marginal zone of spleen also cluster DC strongly. Thus, the ability of B cells to cluster is associated with maturation but also depends on their anatomical site. As most B cells in secondary lymphoid organs have recently crossed endothelium, this event may regulate their ability to cluster DC. We suggest that B cells in blood and lymph express LFA-1 in a low affinity state, and that LFA-1 is activated during extravasation into secondary lymphoid tissues. Integrins are involved in lymphocyte-endothelial binding, and it has been shown that the affinity of rat VLA-4 for VCAM-1 is increased after transendothelial migration (17). Recently, activation of ß2 integrins on naive peripheral lymphocytes has been shown to be induced by the interaction of L-selectin and GlyCAM-1 (18). Quantitative changes in LFA-1 expression are not important, because levels of LFA-1 on B cells from spleen, lymph nodes, and TDL are similar. It is also possible that the expression or affinity of other, unidentified adhesion molecules is up-regulated following transendothelial migration.
In addition to Ag-independent regulation of clustering, B cell adhesion to DC is down-regulated following activation. Thus, naturally activated ex vivo B lymphoblasts or B cells activated by LPS do not adhere to DC. An i.v. injection of LPS strikingly inhibited clustering of B cells from spleen and lymph nodes with L-DC. Intravenous LPS induces migration of splenic B cell from the marginal zones to follicles (19, 20), and the effect of LPS on clustering correlates with the intrasplenic migration of B cells. The effects of LPS on B cells in vivo include partial activation of B cells as expression of ICAM-1, B7, and the transferrin receptor are up-regulated. LFA-1 expression did not change on lymph node B cells and was increased on spleen B cells.
DC increase their ability to stimulate naive T cells during maturation, which correlates with Ag-independent DC/T cell cluster formation. Similar regulation of DC/B cell occurs during DC maturation. DC from spleen and MLN were the most potent in clustering B cells, whereas less mature DC from PP and LP had a lower capacity. L-DC were intermediate. Bone marrow-derived DC bound B cells only weakly. These data reinforce the concept of functional heterogeneity of DC and suggest that physiologic maturation may up-regulate the ability of DC to cluster both T and B cells. In contrast to the increased immunostimulation seen with cultured Langerhans cells (21, 22), all cultured rat DC down-regulated their ability to cluster B cells. The levels of LFA-1, ICAM-1, and OX62 expression did not change on cultured DC, but we were unable to examine ICAM-3 expression.
Little is known of the regulation of DC activity via their surface molecules, although it has been shown that the properties of human and murine DC can be altered by stimulation via CD40 or MHC class II (23, 24, 25, 26, 27). We have shown that cross-linking of MHC class II or Thy-1 but not MHC class I, CD45, CD44, or OX62 has a dramatic stimulatory effect on B cell/DC cluster formation. The effect was not due to simple cross-linking of the two cell types or to FcR interactions, as it occurred with F(Ab')2 fragments and did not occur with Abs to other molecules that are abundant on both cell types (MHC class I and CD45). Also, depletion of Thy-1+ B cells did not inhibit the stimulatory effect of anti-Thy-1 on clustering, formally excluding the possibility that Thy1-stimulated clustering is due to cross-linking. Aggregation was energy, cytoskeleton, and PKC dependent, suggesting the involvement of adhesion molecules. With both anti-MHC class II and anti-Thy-1 Abs, aggregation was neither LFA-1 nor bivalent cation dependent, suggesting involvement of adhesion molecules other than integrins. Taken together, these data demonstrate that cross-linking of surface receptors is a distinct mechanism stimulating B cell/DC cluster formation.
In other systems, cross-linking cell surface molecules may induce cell aggregation. Cross-linking of T cell and B cell Ag receptors (28, 29) stimulates LFA-1/ICAM-1-dependent adhesion. Cross-linking of MHC class II (30, 31) stimulates both LFA-1-dependent and -independent adhesion. Most of these studies have, however, been performed with T and B cells and cell lines.
Conclusions
We suggest that DC/B cell clustering is crucial to the initial stages of a humoral immune response. B cells in the circulatory system have low affinity adhesion molecules, but affinity is up-regulated as they migrate into secondary lymphoid tissue. This up-regulation allows B cells to make short term, LFA-1-dependent interactions with DC in T cell areas. If MHC class II or CD40 on DC has been engaged by T cells recognizing specific peptides, a second, integrin-independent adhesion mechanism is stimulated, allowing for stronger, longer term adhesion of B cells to those DC that are directly involved in T cell activation. The activation of B cells depends on an interaction between them and CD4+ T cells, T cells recognizing MHC class II-peptide complexes on the B cell. Ag processing requires a minimum of 30 min to 1 h (32). Naive B cells enter secondary lymphoid tissues from the blood and thus must acquire Ag in the T cell areas in which they are first activated, either by endocytosis of free Ag or Ag carried by DC.6 The adhesion of B cells to DC involved in the specific activation of T cells allows B cells time to process newly acquired Ag and present it to the T cells being activated by DC. Thus, we conclude that the adhesion of B cells to DC is an integral part of in vivo B cell activation in a primary immune response.
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Acknowledgments |
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Footnotes |
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2 Present address: Leidy Laboratories of Biology, University of Pennsylvania, Philadelphia, PA 19104-6018.
3 Present address: Center for Neurologic Diseases, Brigham and Women’s Hospital, Harvard Medical School, 221 Longwood Ave., Boston MA 02115.
4 Address correspondence and reprint requests to Dr. G. G. MacPherson, Sir William Dunn School of Pathology, South Parks Road, Oxford OX1 3RE, England.
5 Abbreviations used in this paper: DC, dendritic cell; CSP, cytospin; DAB, diaminobenzidine; L-DC, lymph-borne (veiled) dendritic cell; LP, lamina propria; MLN, mesenteric lymph node; MLNX, mesenteric lymphadenectomy; PP, Peyer’s patch; RAM, rabbit anti-mouse; TDL, thoracic duct lymph; XTDL, thoracic duct lymph from a mesenteric-lymphadenectomized rat; LFA, lymphocyte function-associated Ag; GM-CSF, granulocyte-macrophage CSF; PE, phycoerythrin; TCS, tissue culture supernatant; PKC, protein kinase C; MRC, Medical Research Council.
6 M. Wykes and G. G. MacPherson. Dendritic cells present antigen to B cells and modulate isotype switching. Submitted for publication.
Received for publication June 30, 1997. Accepted for publication November 3, 1997.
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 N. Bertho, V. M. Blancheteau, N. Setterblad, B. Laupeze, J. M. Lord, B. Drenou, L. Amiot, D. J. Charron, R. Fauchet, and N. Mooney MHC class II-mediated apoptosis of mature dendritic cells proceeds by activation of the protein kinase C-{delta} isoenzyme Int. Immunol., August 1, 2002; 14(8): 935 - 942. [Abstract] [Full Text] [PDF]
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H. Yoshida, Y. Tomiyama, K. Oritani, Y. Murayama, J. Ishikawa, H. Kato, J.-i. Miyagawa, N. Honma, T. Nishiura, and Y. Matsuzawa Interaction Between Src Homology 2 Domain Bearing Protein Tyrosine Phosphatase Substrate-1 and CD47 Mediates the Adhesion of Human B Lymphocytes to Nonactivated Endothelial Cells J. Immunol., April 1, 2002; 168(7): 3213 - 3220. [Abstract] [Full Text] [PDF]
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 C. Termeer, H. Johannsen, T. Braun, A. Renkl, T. Ahrens, R. W. Denfeld, M. B. Lappin, J. M. Weiss, and J. C. Simon The role of CD44 during CD40 ligand-induced dendritic cell clustering and maturation J. Leukoc. Biol., November 1, 2001; 70(5): 715 - 722. [Abstract] [Full Text] [PDF]
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 K. Takahashi, A. Kenji, T. Norihiro, K. Eisaku, O. Takashi, H. Kazuhiko, Y. Tadashi, and A. Tadaatsu Morphological Interactions of Interdigitating Dendritic Cells with B and T Cells in Human Mesenteric Lymph Nodes Am. J. Pathol., July 1, 2001; 159(1): 131 - 138. [Abstract] [Full Text] [PDF]
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T. Saiki, T. Ezaki, M. Ogawa, K. Maeda, H. Yagita, and K. Matsuno In vivo roles of donor and host dendritic cells in allogeneic immune response: cluster formation with host proliferating T cells J. Leukoc. Biol., May 1, 2001; 69(5): 705 - 712. [Abstract] [Full Text]
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A. Kaser, S. Dunzendorfer, F. A. Offner, O. Ludwiczek, B. Enrich, R. O. Koch, W. W. Cruikshank, C. J. Wiedermann, and H. Tilg B Lymphocyte-Derived IL-16 Attracts Dendritic Cells and Th Cells J. Immunol., September 1, 2000; 165(5): 2474 - 2480. [Abstract] [Full Text] [PDF]
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D. Yang, Q. Chen, S. Stoll, X. Chen, O. M. Z. Howard, and J. J. Oppenheim Differential Regulation of Responsiveness to fMLP and C5a Upon Dendritic Cell Maturation: Correlation with Receptor Expression J. Immunol., September 1, 2000; 165(5): 2694 - 2702. [Abstract] [Full Text] [PDF]
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V. Moulin, F. Andris, K. Thielemans, C. Maliszewski, J. Urbain, and M. Moser B Lymphocytes Regulate Dendritic Cell (Dc) Function in Vivo: Increased Interleukin 12 Production by DCs from B Cell-Deficient Mice Results in T Helper Cell Type 1 Deviation J. Exp. Med., August 21, 2000; 192(4): 475 - 482. [Abstract] [Full Text] [PDF]
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H. Yoshida, Y. Tomiyama, J. Ishikawa, K. Oritani, I. Matsumura, M. Shiraga, T. Yokota, Y. Okajima, M. Ogawa, J.-i. Miyagawa, et al. Integrin-associated protein/CD47 regulates motile activity in human B-cell lines through CDC42 Blood, July 1, 2000; 96(1): 234 - 241. [Abstract] [Full Text] [PDF]
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J. M. Timmerman and R. Levy Linkage of Foreign Carrier Protein to a Self-Tumor Antigen Enhances the Immunogenicity of a Pulsed Dendritic Cell Vaccine J. Immunol., May 1, 2000; 164(9): 4797 - 4803. [Abstract] [Full Text] [PDF]
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F.-P. Huang, N. Platt, M. Wykes, J. R. Major, T. J. Powell, C. D. Jenkins, and G. G. MacPherson A Discrete Subpopulation of Dendritic Cells Transports Apoptotic Intestinal Epithelial Cells to T Cell Areas of Mesenteric Lymph Nodes J. Exp. Med., February 7, 2000; 191(3): 435 - 444. [Abstract] [Full Text] [PDF]
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M. Wykes, A. Pombo, C. Jenkins, and G. G. MacPherson Dendritic Cells Interact Directly with Naive B Lymphocytes to Transfer Antigen and Initiate Class Switching in a Primary T-Dependent Response J. Immunol., August 1, 1998; 161(3): 1313 - 1319. [Abstract] [Full Text] [PDF]
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